ENDOSPERM CULTURE OF COCONUT
A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
IN
HORTICULTURE
MAY 1996
By
Lazarus Agus Sukamto
Dissertation Committee:
Yoneo Sagawa, Chairperson John T. Kunisaki Kenneth Y. Takeda David T. Webb James A. Silva 11
We certify that we have read this dissertation and that, in our opinion, it is satisfactory in scope and quality as a dissertation for the degree of Doctor of Philosophy in
Horticulture.
DISSERTATION COMMITTEE
_ A 0,0xu'Z^ / Chairpej?^on Ill
ACKNOWLEDGEMENT S
Sincere thanks to OTO-BAPPENAS through MUCIA for funding my study, my advisor Dr. Yoneo Sagawa for continuous advice and support. I wish to thank my dissertation committee members,
Drs. David Webb, James Silva, Kenneth Takeda and Mr. John
Kunisaki who gave me support, critical advice and sugges tions. I also wish to thank Dr. Osamu Kawabata for statisti cal help and Dr. Se-Young Kim and other persons for help.
Finally, I am very grateful to my wife Catharina for under standing and caring, my son Leo and my daughter Lei for joy. IV
ABSTRACT
The growth and differentiation of endosperm tissue of coconut cv. Samoan Dwarf in vitro were described. Endosperm tissues produced callus profusely without any enclosing embryo. Explants formed callus three weeks after culture.
Callogenesis occurred in over 95% of all treatments.
There was no significant difference in callogenesis between fruit sources, antipodal and micropylar tissues, 2,4- dichlorophenoxyacetic acid (2,4-D) and 4-amino-3,5,6 - trichloropicolinic acid (picloram) as well with 6 - benzylaminopurine (BAP). Auxin and cytokinin were not necessary to initiate callogenesis.
Growth rate was influenced by fruit source and
concentration of 2,4-D and picloram but not by endosperm region of fruit, two types of auxin (2,4-D and picloram) and
addition of BAP. Growth rate of tissues increased
substantially between 9 to 25 weeks but decreased by 31 weeks of culture. 2,4-D and picloram (lO'^M) inhibited
growth rate in the beginning. The growth rate of the control
was greater than other treatments at 31 weeks after culture.
Tissue (endosperm and callus) browning occurred but did
not inhibit tissue growth. In growth, tissue color changed
from yellowish white, brown to black from which new
yellowish white callus was produced. Callus structure
changed from compact to friable after several transfers. V
Morphogenesis occurred in endosperm callus of antipodal tissue initially treated with lO'^M picloram after 21 weeks
of culture. This "organ" was elongate, opaque and grew
slowly. Its shape changed from triangular with several lumps
on the surface to cylindrical after 14 months. Morphogenesis
also occurred on endosperm callus treated with 207.04xl0'^M
picloram after 17 months.
Histological study of endosperm callus showed
structures which resembled proembryos, embryos with
suspensors, promeristemoids and meristemoids. While "organ
like" structure showed a meristematic layer with a dermal
layer, cortex-like region and central vascular tissue; there
were many small protuberances which resembled embryoids and
shoot with tunica and corpus. Callogenesis and morphogenesis
(shoot organogenesis) occurred in coconut endosperm in
vitro. VI
TABLE OF CONTENTS
Acknowledgements...... iii
Abstract...... iv
List of Tables...... vii
List of Figures...... ix
List of Abbreviations...... xiii
Chapter I. Introduction...... 1
Chapter II. Literature review...... 3 Endosperm culture...... 10 Factors for culture of coconut endosperm...... 12 1. Maturity of explant...... 12 2. Presence or absence of the zygotic embryo....14 3. Culture medium...... 22 4. Growth regulators...... 24 5. Browning...... 32 6 . Culture period...... 35
Chapter III. Initiation of callogenesis...... 37 Introduction...... 37 Materials and methods...... 38 Results and discussion...... 41
Chapter IV. Callus growth and morphogenesis...... 64 Introduction...... 64 Materials and methods...... 65 Results and discussion...... 66
Chapter V. Histological study...... 88 Introduction...... 88 Materials and methods...... 90 Results and discussion...... 92
Chapter VI. General discussion...... 108
Appendix...... 115
References...... 139 Vll
LIST OF TABLES Table page
1. Coconut cultures reported from various tissues...... 5
2. Endosperm cultures reported from various plants...... 15
3. Analysis for percentage of callogenesis after 31 weeks of culture...... 115
4. A summary of the percentage of callogenesis after 31 weeks of culture...... 116
5. Analysis for average tissue browning on the duration of culture...... 117
6 . A summary of browning score of tissues on the duration of culture...... 121
7. Analysis for effect of transfer on growth rate ...... 122
8 . Analysis of growth rate after 9 weeks of culture...... 123
9. Analysis for effect of fruit source on growth rate after 9 weeks of culture...... 124
10. Analysis for effect of 2,4-D and picloram concentrations on growth rate after 9 weeks of culture...... 125
11. Analysis of growth rate after 16 weeks of culture.... 126
12. Analysis for effect of fruit source on growth rate after 16 weeks of culture...... 127
13. Analysis for effect of 2,4-D and picloram concentrations on growth rate after 16 weeks of culture...... 128
14. Analysis of growth rate after 21 weeks of culture.... 129
15. Analysis for effect of fruit source on growth rate after 21 weeks of culture...... 130
16. Analysis for effect of 2,4-D and picloram concentrations on growth rate after 21 weeks of culture...... 131
17. Analysis of growth rate after 26 weeks of culture.... 132
18. Analysis for effect of fruit source on growth rate after 26 weeks of culture...... 133 Vlll
LIST OF TABLES (continued) Table page
19. Analysis for effect of 2,4-D and picloraiti concentrations on growth rate after 26 weeks of culture...... 134
20. Analysis of growth rate after 31 weeks of culture.... 135
21. Analysis for effect of fruit source on growth rate after 31 weeks of culture...... 136
22. Analysis for effect of 2,4-D and picloram concentrations on growth rate after 31 weeks of culture...... 137
23. A summary of growth rate (g per week) of tissues on the duration of culture...... 138 IX
LIST OF FIGURES
Figure page
1. Varied growth and development of endosperm after 4 weeks in control in control. Endosperm tissue was still white, other was brownish and swollen, while others turned dark brown and formed callus...... 52
2. Callus first appeared on the top of the explant, another on the side of the explant which touched the medium after 7 weeks in control...... 52
3. Callus appearances: pale color and slightly friable after 15 months of tissues treated with lO'^M 2,4-D and another with addition of lO'^M BAP ...... 53
4. Tissue (endosperm and callus) browning were varied from little (I = score 1 ) to medium (III = score 2) and high (III = score 3) in control after 21 weeks....53 5. Varied colors of callus and new yellowish white callus grown on the black callus of antipodal tissue treated with 2,4-D and lO'^M BAP after 13 months...... 54
6 . Average browning score of all treatments...... 55
7. Effect of endosperm region on tissue browning...... 56
8 . Effect of 2,4-D and picloram on tissue browning...... 57
9. Effect of 2,4-D and picloram concentrations on tissue browning after 9 weeks of culture...... 58
10. Effect of 2,4-D and picloram concentrations on tissue browning after 16 weeks of culture...... 59
11. Effect of 2,4-D and picloram concentrations on tissue browning after 21 weeks of culture...... 60
12. Effect of 2,4-D and picloram concentrations on tissue browning after 2 6 weeks of culture...... 61
13. Effect of 2,4-D and picloram concentrations on tissue browning after 31 weeks of culture...... 62
14. Effect of lO'^M BAP on tissue browning...... 63
15. Average tissue growth of all treatments...... 74
16. Average growth rate of all treatments...... 75 X
LIST OD FIGURES (continued)
Figure page
17. Growth rate of different fruit sources...... 76
18. Effect of endosperm region on growth rate ...... 77
19. Effect of 2,4-D and picloram on growth rate ...... 78
20. Effect of 2,4-D and picloram concentrations on growth rate after 9 weeks of culture...... 7 9
21. Effect of 2,4-D and picloram concentrations on growth rate after 16 weeks of culture...... 8 0
22. Effect of 2,4-D and picloram concentrations on growth rate after 21 weeks of culture...... 81
23. Effect of 2,4-D and picloram concentrations on growth rate after 2 6 weeks of culture...... 82
24. Effect of 2,4-D and picloram concentrations on growth rate after 31 weeks of culture...... 83
25. Effect of lO'^M BAP on growth rate ...... 84
26. The first appearance of a morphogenesis from antipodal tissue treated with lO'^M picloram after 21 weeks of culture...... 85
27. The "organ" shape became more elongate after 2 months...... 85
28. The "organ" with several lumps on the surface after 8.5 months...... 86
29. The "organ" became a triangular shape after 12 months ...... 86
30. The "organ" became elongate shape after 14 months...... 87
31. Another morphogenesis occurred from tissue treated with 207,04xl0-6M picloram after 17 months ...... 87
32. Endosperm cells of young coconut fruit shows relatively uniform in shape and size, stained with toluidine blue ...... 96 XI
LIST OF FIGURES (continued)
Figure page
33. Nuclei of endosperm cells consisted of 4 - 5 nucleoli, unstained...... 96
34. Cells of endosperm callus shows dark and light areas, stained with acid fuchsin and toluidine b lu e ...... 97
35. Enlargement of dark area of Fig. 34. Cells with a lot of lipid droplets...... 97
36. Enlargement of light area of Fig. 34, cells underwent many divisions with few lipid droplets and long nucleolus appearance...... 98
37. Various round cell clusters with 3 - 5 cells of coconut endosperm treated with 10'^ 2,4-D, stained with IKI ...... 98
38. Formation of linear cells structure, stained with acid fuchsin and toluidine blue ...... 99
39. Structure resembles a young embryo with suspensor of coconut endosperm treated with lO'^M 2,4-D, stained with IKI...... 99
40. Formation of promeristemoid with 7 cells cluster, stained with Feulgen-fast green...... 100
41. Formation of meristemoid with 9 cells cluster, showing different types of divisions, stained with P A S ...... 100
42. Meristemoid consisting over 15 cells cluster of coconut endosperm treated with lO'^M 2,4-D and BAP, stained with IKI ...... 101
43. Meristemoid consisting over 20 cells cluster of coconut endosperm treated with 10'^ 2,4-D and BAP, stained with Feulgen-fast green...... 101
44. Central vascular tissue of "organ-like" structure, showing parenchymatous cortex and dermal layer, stained with toluidine blue...... 102
45. Sheating base of the cotyledon and vascular tissue toward stem tip of "organ-like" structure, stained with toluidine blue ...... 102 Xll
LIST OF FIGURES (continued)
Figure page
46. Enlargement of vascular tissue showed tracheids of xylem, stained with toluidine blue...... 103
47. Meristematic periphery of "organ-like" structure, stained with toluidine blue ...... 103
48. Meristematic mantle of "organ-like" structure, stained with toluidine blue...... 104
49. Protuberances of "organ-like" structure, stained with toluidine blue ...... 104
50. Protuberances of "organ-like" structure, stained with toluidine blue...... 105
51. Protuberances of "organ-like" structure, stained with toluidine blue...... 105
52. Protuberance of "organ-like" structure, stained with toluidine blue ...... 106
53. Formation of embryoid of "organ-like" structure, stained with toluidine blue...... 106
54. Formation of one layer of tunica and cell group of corpus of "organ-like" structure, stained with toluidine blue ...... 107 Xlll
ABA abscisic acid
AC activated charcoal
Ads adenine sulfate
AZI 7-azaindole
BAP 6 -benzylaminopurine
BM basal medium
CH casein hydrolysate
CW coconut water
2,4-D 2 ,4-dichlorophenoxyacetic acid
DAP days after pollination
EC embryogenic cells
GA gibberellic acid
GLM General Linear Model
HCHO paraformaldehyde
HR historesin lAA indole-3-acetic acid lAA-ala lAA-L-alanine lAA-asp lAA-L-aspartic acid
I BA indole butyric acid
IKI potassium iodide-iodine
2iP 6_ ('y-'y-dimethylallylamino) purine
I PA indole-3-propionic acid
KN kinetin
MS Murashige & Skoog
ME malt extract XIV
LIST OF ABBREVIATIONS (continued)
NAA a-naphthaleneacetic acid
NEC nonerobryogenic cells
NOA 2-naphthoxy acetic acid
PAS periodic acid Schiff
PCMP 2(p-chlorophenoxy) 2-methylpropionic acid
Picloram 4-amino-3,5,6 -trichloropicolinic acid
PPM part per million
RCBD randomized complete block design
SAS Statistical Analysis System
SE somatic embryogenesis
TIBA 2,3,5-triiodobenzoic acid
YE yeast extract
W White woe weeks of culture CHAPTER I
INTRODUCTION
Coconut (Cocos nucifera L.) belongs to the Arecaceae family and is distributed in tropical areas. There are approximately 11 million ha of coconuts growing in more than
80 countries of the tropics (Thangaraj and Muthuswami, 1990;
Persley, 1992). Most belong to small holders who grow them for domestic use (Benbadis, 1992). The various common names given to coconut reflect its usefulness, e.g. tree of life, tree of abundance, tree of heaven (Green, 1991). All parts of the plant are very useful for humankind (Menon and
Pandalai, 1957; Reynolds, 1982; Blanton and Blake, 1983b;
Thangaraj and Muthuswami, 1990; Green, 1991; Persley, 1992).
The plants are also used as an ornamental plant in regions such as Hawaii and Florida.
The plant is traditionally propagated by seed which is recalcitrant and has a short storage life. The plant is
long-lived but has a very long juvenile phase. It is generally cross pollinated and very heterozygous (Tammes and
Whitehead, 1969; Reynolds, 1982; Blake, 1983; Bhaskaran,
1985; Karunaratne and Periyapperuma, 1989). Therefore vegetative propagation is needed in order to have uniform plants.
Although micropropagation in vitro from various tissues 2
(embryo, stem, leaf, root, inflorescence, endosperm, and anther) have been attempted, success is mostly limited to immature unselected plants producing very few plants (Brack- pool et al,, 1986; Pannetier and Buffard-Morel, 1986; Blake,
1991). Micropropagation of other palms, oil and date palms, has been successful (Tisserat, 1979; Corley et al., 1981;
Sharma et al., 1984; Rao and Ganapathi, 1993). A reliable method of clonal propagation of coconut would be a great asset for breeding program due to fast multiple of selected plant in relatively short time (Blake, 1991).
Coconut endosperm provides large and uniform explant tissue for experimentation. Micropropagation of endosperm tissue would be advantageous since explants come from mature selected plants. Plants from endosperm may be triploids and may produce fruits without embryos or produce no fruit. If they bear no fruit, it will have a secondary advantage for
Hawaii and other areas where it is used for ornamental purposes since large fruits must be removed to avoid the dangers encountered by falling fruits.
The objectives of this study are twofold; first, to establish a protocol for initiation in vitro of callogenesis from endosperm of mature selected plants and second, to induce morphogenesis and ultimately plant regeneration. CHAPTER II
LITERATURE REVIEW
Some methods of vegetative propagation have been applied in coconut. Airlayering of tall trees by attaching a wooden box filled with sand or coir dust on the wounded stem, results in a rooted tree that can be planted. This reduces tree height and increases production (Davis, 1969).
Airlayering of bulbils emerging abnormally within the spathe instead of an inflorescence produces only a limited number of plants. These plants at maturity are sterile because only bulbils instead of inflorescence are produced (Davis, et al., 1985). Splitting the growing point of stem results in more than one shoot (Davis, 1969; Fisher and Tsai, 1979).
None of these methods is satisfactory. Thus there is a need to pursue in vitro multiplication of coconut.
In vitro studies of coconut started in the early 1950's with embryo culture (Cutter and Wilson, 1954). Researchers in the Philippines regenerated plants from zygotic embryos of "Makapuno" cultured in vitro (Balaga and De Guzman,
1971; De Guzman et al.,1971; De Guzman and Manuel, 1977) and in Indonesia from embryos of "Kopyor" which is similar to
"Makapuno" (Tahardi, 1987b). "Makapuno" or "Kopyor" has a gelatinous endosperm which is highly valued as a delicacy in drinks or ice cream (Tammes and Whitehead, 1969). Although 4 the embryo is fully developed, it cannot be grown from seed due to disfunction of the endosperm (Tammes and Whitehead,
1969; De Guzman, 1981).
Plants of other coconuts have also been propagated through embryo culture (De Guzman et al., 1978; D'Souza,
1982; Gupta et al., 1984; Bah, 1986; Bhalla-Sarin et al.,
1986, and Sugimura et al., 1988). Tahardi (1987a) reported that only roots grew from sliced embryos of coconut.
Plantlets were regenerated through callogenesis and embryogenesis from 6 - 7 month-old coconut embryos iji vitro
(Karunaratne and Periyapperuma, 1989).
Others reports of in vitro studies of coconut include cultures of cotyledon (Jagadeesan and Padmanabhan, 1982), stem apices (Blake and Eeuwens, 1982; Branton and Blake,
1983a; Gupta et al., 1984), leaves (Pannetier and Buffard-
Morel, 1982a; Raju et al., 1984; Verdeil et al., 1989;
Buffard-Morel et al., 1992), inflorescences (Blake and
Eeuwens, 1980; Branton and Blake, 1983a; 1986; Gupta et al.,
1984; Sugimura et al., 1988; Sugimura and Salvana, 1989;
Verdeil et al., 1989), endosperms (Fisher and Tsai, 1978;
Bhalla-Sarin and Bagga, 1983; Kumar et al., 1985), roots
(Bhalla-Sarin and Bagga, 1983; Schwabe, 1983) and anthers
(Thanh-Tuyen and De Guzman, 1983; Monfort, 1985). Explants from these tissues, especially leaves and inflorescences produced embryoids and plantlets. Table 1 is a summary of coconut cultures reported from various tissues. Table 1. Coconut cultures reported from various tissues.
Explant Growth regulator Response Reference source
Embryo 57.08-114.16xlO*M lAA shoot and root De Guzman et al., 1971
0.54xl0-*M NAA plantlets De Guzman et al., 1978
26.85-40.28xlO-*M NAA callus D'Souza, 1982
without bud-like structure
5.37-10.74xl0*M NAA root and pneumatophores
12-20x10-^ 2,4-D callus Karunaratne and Periyapperuma, 1982 2-8xlO-*M 2,4-D + lO^M BAP and KN embryoids and each plantlets 8.12x10-^ lAA-ala or 6.89xlO-*M callus Bhalla-Sarin and lAA-asp Bagga, 1983
6.89xlO‘M lAA-asp + 9.29x10-^ KN shoot and root or 2.69xlO*M NAA + 8.87xlO-*M BAP
2.26-22.6xl0‘M 2,4-D + 0.05- shoot and Gupta et al., 1984 5.37x10-^ NAA + 0.44-4.44x10-^ BAP root
tji Table 1. Coconut cultures reported from various tissues (continued).
Explant Growth regulator Response Reference source
Embryo without shoot and root Karunaratne et al., 1985
without shoot and root Bah, 1986 10.74xl0*M lAA-asp or 8.12xl0'^ callus Bhalla-Sarin et al., lAA-ala 1986
6.89xlO*M lAA-asp + 9.29xlO-*M KN plantlet or 10.74xl0*M NAA without shoot and radicle Tahardi, 1987b
57.08xl0-*M lAA shoot and root 452.50xl0*M 2,4-D +BAP + 2iP callus and root Sugimura et al., 1988 like structure
100-300x10-^ NAA shoot and root Ashburner et al., 1993
Cotyledon 4.44xlO-*M BAP + 2.69x10'^ NAA callus Jagadeesan and Padmanabhan, 1982 4.44xlO'*M BAP + 5.37xlO-*M NAA rootlets and pneumatophores
o> Table 1. Coconut cultures reported from various tissues (continued).
Explant Growth regulator Response Reference source
Stem 100'‘-10-'M 2,4-D plantlets Blake and Eeuwens, apice 1982 2.5xlO‘’M 2,4-D + AC or 2.5xlO‘M roots 2 , 4-D 1 0 ‘’M 2,4-D + 5x10-^ BAP + SxlO"^ callus Branton and Blake, 2iP 1983a
10*M 2,4-D + 5x10-^ BAP + 5x10'^ embryoids 2iP 4.52xlO“M 2,4-D callus Gupta et al., 1984
0.27xl0*M NAA + 2.22xlO-*M BAP + embryo-like 0.9 3xl0iyi KN structure Leaf 2.26xlO-^M 2,4-D embryo-like Gupta et al., 1984 structure KN, 2iP, BAP + NAA, lAA, IPA callus and Raju et al., 1984 embryoids withdrawal NH^, HCHO, CH, reduced auxin to 75% and increased CK to plantlets 125% auxin callus Verdeil et al., 1989 reduce gradually auxin embryoids and plantlets Table 1. Coconut cultures reported from various tissues (continued).
Explant Growth regulator Response Reference source
Leaf 1.3 5-2.70X10''M 2,4-D embryo-like and Buffard-Morel et 1 shoot and al., 1992 0.22-0.45xl0'"M 2,4-D root
Inflores 2.5xlO-'M NAA -I- 5xlO*M BAP to shootlet and Blake and Eeuwens, cence 2.5xlO-‘-2.5xlO-^M NAA -I- 5xlO*M BAP root 1980 lO^M 2,4-D -I- 5xlO*M 2iP 5xlO*M callus Branton and Blake BAP -f- 2.7x10-^ AdS 1983a; 1986
10'*M 2,4-D -f 5xlO"M 2iP -f 5xlO'*M embryoids and BAP -I- 2.7x10-^ AdS 2 plantlets 4.52xlO-''M 2,4-D callus Gupta et al., 1984
2.3xlO'^M 2,4-D root and shoot like structure
90.50-109xl0iy[ 2,4-D + BAP -I- 2iP callus Sugimura et al., 1988; Sugimura and 4.52-22.62xl0-‘M 2,4-D -I- BAP + 2iP shoot-like Salvana, 1989 structure or root
auxin callus Verdeil et al., 1989 reduce gradually auxin embryoids
CO Table 1. Coconut cultures reported from various tissues (continued).
Explant Growth regulator Response Reference source
Endosperm 10.74xl0'^ NAA + 9.29xlO‘M KN no Bhalla-Sarin and Bagga, 1983
5.71x10-^ lAA + 4.65xlO"M KN callus Fisher and Tsai 1978
10.7-26.8xl0-*M NAA continue growth 2.3xlO“'M 2,4-D + 9.3xlO*M KN callus Kumar et al., 1985
4.5x10-^ 2,4-D + 2.3xlO*M KN continue growth 9.05x10'^ 2,4-D or 9.80xl0«M IBARoot roots or Schwabe, 1983 callus
6.89xlO*M lAA-asp + 8.87xlO*M embryoids Bhalla-Sarin and BAP Bagga, 1983 Anther 4xlO"M TIBA embryo Monfort, 1985
10.74xl0-"M NAA embryo Thanh-Tuyen and De Guzman, 1983
VD 10
ENDOSPERM CULTURE
Polyploid heterosis has occurred in apples, pears.
Citrus and grapes and results in greater vigor, larger fruit size and generally greater yield (Sanford, 1983). In Petunia axillaris triploids were more vigorous and had bigger flowers than the diploid or tetraploid plants (Gupta, 1982).
Populus tremuloides triploids were superior for wood production compared to diploid counterparts (Johri et al.,
1980) .
Triploid plants have been produced by crossing diploid and tetraploid plants in Citrus (Soost and Cameron, 1980;
Oiyama and Kobayashi, 1990; Oiyama et al. 1991) and in papaya (De Zerpa, 1957) but seed germination was very poor.
Survival was low due to the failure of endosperm development and subsequent abortion of embryo which correlates with the ploidy ratio of endosperm to embryo that is not exact 3 : 2 as in vivo (Sita, 1987; Soost, 1987; Grosser, 1994).
Producing triploid plants by crossing method is cumbersome since it takes a long time for economical purpose; an alternative is to regenerate plants from endosperm explants in vitro (Sita, 1987).
Endosperm is triploid in over 81% of flowering plants.
It is the result of fusion of two polar nuclei of female gametophyte and one of male gametes (Johri et al., 1980).
Therefore, it differs genotypically from the embryo in gene- 11 dosage (Kovoor, 1981). Endosperm is a fairly homogeneous mass of parenchymatous cells and lacks vascular elements
(Johri and Bhojwani, 1977; Johri et al., 1980). However, cells can vary in size and ploidy (Abraham and Mathew,
1963) .
The function of endosperm is to nurture the embryo during its heterotrophic phase of growth and to provide combustible sources of energy during seed germination (Johri and Bhojwani, 1977; Raghavan, 1986). Endosperm may be consumed entirely by the embryo so that mature seed is called nonendospermous. If it persists in mature seed as a massive tissue, it can store reserve food materials in the form of starch, fat or protein and is called endospermous
(Johri and Bhojwani, 1977; Johri et al., 1980; Bhojwani,
1984) .
Endosperm of angiosperms can grow and differentiate into organs (Nag and Johri, 1971; Bhojwani and Razdan,
1983). Triploids from culture may be superior to those from crossing tetraploid and diploid plants, due to the genetically 'unreduced' nature of the 2 n polar fusion nucleus in the central cell of the megagametophyte (Knight and Alston, 1969).
Endosperm culture produced shoots or plantlets in
Ricinus communis. Exocarpus cupressiformis. Actinidia chinensis. Codiaeum varieaatum. Jatropha panduraefolia.
Putraniiva roxburghii, Orvza sativa, Dendrophthoe falcata. 12
Scurrula pulverulenta. Taxillus vestitus. Prunus persica.
Pvrus malus. Jualans regia. Citrus grandis. C. sinensis and
Santalum album (Rangaswamy and Rao, 1963; Satsangi and Ram,
1965; Johri and Bhojwani, 1977; Bhojwani, 1984; Tulecke et al., 1988; Chen et al. 1990). Thus it may be possible to regenerate coconut plants from cultured endosperm.
The endosperm of coconut is nuclear type with large endospermous seed which endosperm remains until germination
(Johri et al., 1992). The endosperm is free of microorganisms (Fernandez, 1988). Even though in vitro culture of adult tissue of coconut is recalcitrant
(Reynolds, 1982; D'Souza et al., 1983), regeneration is possible (Branton and Blake, 1983a; 1986; Verdeil et al.,
1989). Cultures required high auxin levels for callus initiation, with gradually decreasing auxin concentrations and cytokinin for somatic embryogenesis (SE) induction
(Blake, 1983; Verdeil et al., 1994).
FACTORS FOR CULTURE OF COCONUT ENDOSPERM
1. Maturity of explant
The age of the endosperm at the time of culture is critical for growth in vitro (Nag and Johri, 1971). Explants 13 from younger fruits responded better to culture (Iyer, 1982;
Hanower and Pannetier, 1982; Tisserat, 1984; Aitken-Christie et al., 1985; Paranjothy, 1986b; Karunaratne and
Periyapperuma, 1989; Karunaratne et al., 1991).
Coenocytic (free-nuclear) endosperm did not survive in culture because of lack of starch (Srivastava, 1982).
Endosperm of 6 - 7 months postanthesis in coconut (Kumar et al., 1985), in which solid endosperm started to form in the antipodal end of coconut fruit (Tammes and Whitehead, 1969), was responsive to culture.
Maize endosperm taken between 8 to 12 days after pollination (DAP), grew in vitro when cells were meristematic (Straus and LaRue, 1954; Sternheimer, 1954;
Tamaski and Ullstrup, 1958). Endosperm of rye grass responded in culture when excised 9-10 DAP (Norstog,
1956), for rice 4 - 7 DAP (Nakano et al., 1975). Mature rice endosperm also gave positive results (Bajaj et al., 1980).
Young endosperm (celliferous stage) was responsive to culture in Citrus grandis and apple (Wang and Chang, 1978;
Mu and Liu, 1978). Explants removed 7-10 DAP in cucumber
(Nakajima, 1962), 56 DAP in walnut (Tulecke, et al., 1988) and 98 - 119 DAP in Citrus sinensis cv. Hongjiang (Chen, et al. 1990) were responsive to culture.
Mature endosperms in Exocarpus cupressiformis.
Leptomeria acida, Niaella damascena. Nuvtsia floribunda.
Osvris wightiana. Scurrula pulverulenta. Phoradendron 14 tomentosum. Dendrophtoe falcata. Taxillus cunefatus. T. vestitus. Leptomeria acida. Croton bonplandianum. Santalum album. Ricinus communis. Jatropha panduraefolia. Putranjiva rpxburghii, Sapium sebiferum. Coffea arabica. Annpna squamosa. Achras. apple, parsley and pecan were responsive to culture (Johri and Bhojwani, 1965; Nag and Johri, 1971,
Srivastava, 1973; 1982; Sethi and Rangaswamy, 1976; Bajaj et al., 1980; Cheema and Mehra, 1982; Nair et al., 1986, Sita,
1987). Table 2 is a summary of endosperm cultures reported from various plants.
2. Presence or absence of the zygotic embryo
Enclosure of the embryo in the explant was essential for callus initiation in endosperm culture of Dendrophthoe.
Exocarpus. Jatropha. Phoradendron. Putrani iva. Scurrula. and
Taxillus (Reinert et al., 1977). Mature endosperm of Croton.
Ricinus and Putrani iva without enclosed embryos failed to proliferate in vitro (Srivastava, 1982). Mature endosperm of
Annona squamosa produced only callus when cultured after radicle emergence 2 - 4 days (Nair et al., 1986). In Citrus sinensis endosperm which had an enclosed embryo for 2 days responded best to culture while failing after 8 days (Chen et al., 1990).
Nag and Johri (1971) reported that an enclosed embryo was not necessary for initiation of callus proliferation of endosperm of 5 parasitic taxa: Dendrophthoe falcata. Nuytsia Table 2. Endosperm cultures reported from various plants.
Plant stage Supplements Response Reference
Maize 12 DAP 5000 ppm yeast extract (YE) callus Straus and LaRue, 1954
10-12 2 0 % clear tomato juice callus Sternheimer, DAP 1954
8-11 5000 ppm YE callus Tamaski and DAP Ullstrup, 1958
9.05xl0‘‘^M 2,4-D callus Zhu et al., 10-12 1988 DAP without plantlets Rye grass 9-10 DAP 0.25-0.50% YE + 5.71xlO’^M lAA callus Norstog, 1956 Cucumber 7-10 DAP 5000 ppm YE callus Nakajima, 1962 Exocarpus mature 5.71xlO'‘^M lAA + 4.65xlO’'^M KN callus, Johri and cupressiformis + 400 ppm casein hydrolysate shoot or Bhojwani, 1965 (CH) root Osvris mature 22.62xlO"'^M 2,4-D + 500 ppm CH callus Johri and wiqhtiana Bhojwani, 1965 11.42xlO'^M lAA + 23.23xlO''^M vascularized KN outgrowth Putrani iva mature 9.05x10'^ 2,4-D + 23.23xlO'^M callus Johri and roxburqhii KN + 2500 ppm YE Bhojwani, 1965
11.42X10''^M lAA + 23.23xlO'^M callus Srivastava, KN + 1000 ppm CH 1973 1.2xlO'^M lAA + 2.4xlO'^M 2iP shoots oi Table 2. Endosperm cultures reported from various plants (continued).
Plant stage Supplements Response Reference
Nuvtsia mature 24.60xl0‘^M IBA + 23 . 23xlO''^M callus Nag and floribunda KN + 2000 ppm CH Johri, 14.27xlO'^M lAA + 23.23xlO‘'^M tracheid- 1971 KN + 2000 ppm CH like cells Dendrophthoe mature 24.60xl0'^M IBA + 23 . 23xlO‘*M callus Nag and falcata KN + 2000 ppm CH Johri, 14.27xlO'^M lAA + 23.23xlO'^M shoots and 1971 KN + 2000 ppm CH haustoria Taxillus mature 24.60xl0'^M IBA + 23.23xlO’‘^M callus Nag and cuneatus KN + 2000 ppm CH Johri, 14.27xlO‘^M lAA + 23.23xlO''^M shoot buds 1971 KN + 2000 ppm CH Leptomeria mature 24.60xl0‘‘^M IBA + 23 .23xlO'^M callus Nag and acida KN + 2000 ppm CH Johri, 14.27xlO''^M lAA + 23.23xlO''^M shoots 1971 KN + 2000 ppm CH Ricinus mature 22.62xlO'*^M 2,4-D + 9.29xlO’^M callus Satsangi and communis KN Ram, 1965 22.62xlO’'^M 2,4-D + 9.29xlO‘^M embryoids KN + 2500 ppm YE11.42xlO''^M
lAA + 23.23X10‘^M KN + 1000 callus with Johri and ppm CH or tracheidal Srivastava, 9.05xl0'*^M 2,4-D + 23.23xlO‘'^M cells and 1972 KN + 2500 ppm YE vascular bundle-like
CTv Table 2. Endosperm cultures reported from various plants (continued).
Plant stage Supplements Response Reference
Croton mature 9.05x10'% 2,4-D + 23.23x10'% callus Bhojwani and bonplandianum KN + 2500 ppm YE Johri, 1971 10'% lAA, IBA or NAA roots Jatropha mature 9.05x10'% 2,4-D + 23.23x10'% callus Johri and panduraefolia KN + 2500 ppm YE Bhojwani, 1965
9.05X10'% 2,4-D + 23.23x10'% callus Srivastava, KN + 2500 ppm YE 1971 0.54x10'% NAA + 2.3 2x10'% KN shoots and + 500 ppm CH roots
Scurrula mature 5.71x10'% lAA + 400 ppm CH callus Bhojwani and pulverulenta Johri, 1970 5.71x10'% lAA + 4.65x10'% KN shoot and + 400 ppm CH haustoria Taxillus mature 2X10'% lAA, IBA, NAA, 2,4-D or callus Johri and vestitus 2,4,5-T Nag, 1970 2x10'% KN, BAP or 2iP shoot buds 24.60x10'% IBA + 23.23x10'% callus Nag and KN + 2000 ppm CH Johri, 1971 14.27x10'% lAA + 23.23x10'% shoots and KN + 2000 ppm CH haustoria
Niqella mature 5xl0'%-10'% 2,4-D callus and Sethi and damascena embryoids Rangaswamy, 1976 Table 2. Endosperm cultures reported from various plants (continued).
Plant stage Supplements Response Reference
Parsley mature without callus, Masuda et shoot and al., 1977 root Citrus grandis cellular 9.05xl0'^M 2,4-D + 22.19xlO’‘^M callus Wang and cv. Bei-pei and stage BAP + 1000 ppm CH Chang, 1978 C. sinensis cv. 5.77-43.32xl0‘^M GA embryoids Chin-cheng and plantlets
C. sinensis cv. 98-119 2.26x10’'^ 2,4-D + 2.22xlO'^M callus Chen et Hongj iang DAP BAP + 1000 ppm CH al., 1990
0. 29-14. 43xlO’'^M GA root and shoot
C. sinensis cv. 84-98 DAP 9.05xl0'^M 2,4-D + 22.19xlO'^M callus emitter et Ridge Pineapple BAP + 23.23x10'^ KN + 1000 ppm al., 1990 CH + 500 ppm ME l.llxlO''^M BAP + 14.8x10''^ Ad embryos, + 5.77xlO'^M GA + 500 ppm CH root or shoot
C. grandis cv. 84-98 DAP same medium callus and emitter et White Siamese embryoids al., 1990
and C. Xparadisi 84-98 DAP same medium callus emitter et cv. Duncan al., 1990
03 Table 2. Endosperm cultures reported from various plants (continued).
Plant stage Supplements Response Reference
Sandalwood 0.6-0.8 cm 4.52-9.05xl0'^M 2,4-D or + callus Sita et al., green 2.22-8.87xl0’‘^M BAP + 5.37x10' 1980 fruit •^M NAA 5.71X10‘‘^M lAA + 1.33xlO'^M BAP embryoids + 1.39xlO'^M KN + 2.89X10'‘^M GA
2.85xlO'‘^M lAA shoot and root Walnut young and 2,4-D + 4.65xlO‘‘^M KN + 500 ppm callus Cheema and mature CH Mehra, 1982 1.07x10'*^ NAA + 2000 ppm CH roots
without shootbud like
cv. Manregian 56 DAP 0.06x10'^ IBA + 4.44xlO'^M BAP embryos Tulecke et + 9.2 9xlO'^M KN al., 1988
without shoot and root Pecan cv. mature 2,4-D + 4.65xlO'‘^M KN + 500 ppm callus Cheema and Mahan CH Mehra, 1982 1.07x10'^ NAA + 2000 ppm CH roots Pear cv. cellular 2.26X10'^M 2,4-D + 2.85xlO''^M callus Zhao, 1988 Jinfeng stage lAA + 6 .6 6 xlO'^M BAP 8.87xlO‘^M BAP + 28.89xlO'‘^M GA shoots
8.57xlO'^M lAA shoot + root >Xl Table 2. Endosperm cultures reported from various plants (continued).
Plant stage Supplements Response Reference
Rice 4-7 DAP lO'^M 2,4-D + 0.4% YE or callus Nakano et + 5x10'% KN al., 1975
lO'^M lAA + 0.4% YE or shoot 5xlO'^M KN + + 0.4% YE buds
4-8 DAP 9.05xl0‘*^M 2,4-D callus Bajaj et and mature al., 1980 22.84X10’^M lAA + 9.29xlO'^M KN shoots and haustoria tomato 21 DAP- 4.52xlO''^M 2,4-D + 0.44xl0’‘^M callus Kagan-Zur mature BAP + 28.89xlO’^M GA et a l . , 1990 Apple cv. King- young 2.26x10'^ 2,4-D + 4.44x10'^ callus Mu and Liu, kuang BAP + 500 ppm CH 1978
Annona squamosa mature 5.37xlO'^M NAA+ 0.88xl0‘^M BAP + callus Nair et 0.46x10'^ KN + 2.89xlO'^M GA al., 1986
28.54xlO''^M lAA root
2.69x10'^ NAA + 8.87xlO’^M BAP shoot Loquat 35-60 DAP 2,4-D, NAA and BAP callus and Chen et embryoids al . , 1988a
NAA and Zeatin plantlets
to o Table 2. Endosperm cultures reported from various plants (continued).
Plant stage Supplements Response Reference
Lvcium barbarum 20 DAP 0.45xl0'^M 2,4-D callus Wang et al. , 1988 0.88xl0’*M BAP plantlets Actinidia cellular 2.26xlO‘^M 2,4-D or O.BOxlO'^^M callus Gui et al., chinensis stage NAA + 13.68xlO'^M Zeatin 1988
4.56xlO‘*^M Zeatin plantlets or buds Actinidia 101-128 13.7xlO'^M Zeatin + 0.54xl0'^M callus and Mu et a l . , interspecific DAP NAA + 400 ppm CH buds 1990 hybrids 23xlO‘‘^M Zeatin + 1.7xlO‘‘^M lAA plantlets + 400 ppm CH
to 22 floribunda. Taxillus cuneatus. T. vestitus and Leptomeria acida. Sita (1987) successfully cultured endosperm from fresh green fruits of sandalwood with or without embryos.
Proliferation in endosperm cultures of Actinidia (Mu et al.,
1990), Citrus qrandis. C. sinensis (Wang and Chang, 1978), pear (Zhao, 1988), pecan and walnut (Cheema and Mehra, 1982) occurred without enclosed embryo.
In Croton bonplandianum. Putraniiva and Citrus, the requirement for enclosed embryo was replaced by soaking endosperm pieces in 1 - 2 mg*!’^ gibberellic acid (GA)
(Johri and Bhojwani, 1977;
Srivastava, 1982; Nair et al., 1986) or 1 mg*l‘’ zeatin
(Sita, 1987).
Fisher and Tsai (1978) successfully produced callus from a single explant of young coconut cv. Golden Malayan
Dwarf endosperm without any indication of an enclosed embryo. Bhalla-Sarin and Bagga (1983) failed to induce callogenesis of 8 - 12 month old endosperm of coconut cv.
West Coast Tall, Dwarf and Laccadive with enclosed embryo.
Kumar et al. (1985) were able to induce a subculturable callus from 6 - 7 month old endosperm of coconut cv. West
Coast Tall with enclosed embryos.
3. Culture medium
White (W) or Murashige and Skoog (MS) media are
commonly used and MS medium is more frequently used because 23 it contains higher content of inorganic salts and nitrogen
(Sharp et al., 1980; Bhojwani, 1984; Chen et al., 1988).
Eeuwens (1976) reported that a new mineral formulation
(Y3) was better than W or MS medium for micropropagation of coconut due to mineral deficiencies in macro elements such as nitrogen (ammonium), potassium, phosphate and in micro elements such as iron, iodine, molybdenum in W medium and MS is deficient in iodine. The addition of glutamine, arginine, asparagine and sucrose to Y3 medium increased fresh and dry weight of coconut callus (Eeuwens, 1978).
Gelrite or phytagel gelling agent is a complex of extracellular polysaccharides produced by bacteria
Pseudomonas elodea. It is composed of glucuronic acid, rhamnose and glucose and is exceedingly clear. Phytagel has
less free minerals and organic impurities than agar (Kyte,
1987; Bonga and Von Aderkas, 1992; Sigma, 1993).
Tissue growth was better on phytagel than agar due to
stimulative substances and lack of impurities in culture of
coconut (Sugimura et al., 1988), bamboo (Huang and
Murashige, 1983), mango (DeWald et al., 1989a) and cucumber
(Ladyman and Girard, 1991). Phytagel (1.7 - 1.75 g*l’^) was more effective for proliferation of globular and SE of
longan and mango (Litz, 1988; DeWald et al., 1989a; 1989b).
Liquid medium is sometimes best for certain culture
stages. Liquid static medium was better than agar for
initiating coconut callus (Blake and Eeuwens, 1982). Stirred 24 liquid medium improved the growth rate of oil palm callus
(Lioret, 1982).
Age of medium containing 2,4-D influenced growth response of coconut cultures. Explants showed minimal growth and died on one day-old medium, while a better response occurred with five day-old medium and the best response on nine day-old medium due to 2,4-D concentration (Ebert and
Taylor, 1990).
4. Growth regulators
Whole plants are autonomous with regards to growth regulators but isolated tissues or cells require auxins or cytokinins to initiate and maintain growth until they become habituated or organized (Everett, et., 1978). Morphogenesis
in vitro can be regulated by growth regulators (Christianson and Warnick, 1983). Skoog and Miller (1957) found that the balance of auxin and cytokinin in culture medium governed organogenesis. Auxins induced callogenesis, adventitious plantlets and roots in palms (Tisserat, 1979a; Paranjothy and Rohani, 1982; Reynolds, 1982; Paranjothy, 1986a; 1987).
Among auxins, 2,4-D was the most effective when compared to indole acetic acid (lAA), 2-naphthoxy acetic acid (NOA), a-naphthalene acetic acid (NAA), or indole butyric acid (IBA) in coconut culture (Blake and Eeuwens,
1982; Pannetier and Buffard-Morel, 1986; Karunaratne and
Periyapperuma, 1989). Bhalla-Sarin et al. (1986) reported 25 that lAA-conjugates (lAA-asp and lAA-ala) induced callus and differentiation of coconut embryos.
Auxin at high concentrations (10‘^M - lO'^M) was necessary for callus induction in palm, especially on medium supplemented with 1 - 3 g*l'^ activated charcoal (AC)
(Karunaratne and Periyapperuma, 1989; Branton and Blake,
1983a; Sugimura and Salvana, 1989; Tisserat and DeMason,
1980; Kumar et al., 1985; Jesty and Francis, 1992; Reynolds and Murashige, 1979; Sharma et al., 1984; Zaid and Tisserat,
1984; Gupta et al., 1984; Blake and Eeuwens, 1982).
For mass propagation, somatic embryogenesis (SE) is preferable to organogenesis since it produces integral shoot and root meristems, a large number of plants, and it is also better for genetic engineering and encapsulation as artificial seeds (Gupta, 1987; Sita, 1987; Chen et al.,
1988) .
Embryogenesis begins in callus cultures with a single cell which differs from neighbouring vacuolated parenchymatous cells by having dense cytoplasm, starch accumulation and it is often surrounded by a thickened cell wall. Internal divisions of the small densely cytoplasmic cells form proembryos and can lead to the development of globular, heart shaped and torpedo shaped embryos which can be converted into plants (Kohlenbach, 1978).
Undefined primordia are initiated in disorganized callus. These may be shoot and/ or root primordia or even 26 suppressed somatic embryos ["proembryogenic masses" (PEM)]
(Wernicke and Milkovits, 1986).
Manipulating the concentration of hormones resulted in
SE maturation (Bhaskaran, 1985). 2,4-D is usually essential for culture establishment but is usually removed or its concentration is lowered for growth and differentiation
(Murashige, 1974). Low concentrations of "weak" auxins such as NAA or IBA together with low concentrations of cytokinins such as BAP or KN promoted shoot development of oil palm embryos, while roots were obtained in liquid media after brief exposure to NAA (Paranjothy and Rohani, 1982).
The use of auxins other than 2,4-D can overcome the failure of some callus cultures to differentiate due to the presence of residual 2,4-D (Murashige, 1974; Brown and
Charlwood, 1990). Another auxin with properties similar to
2,4-D is 4-amino-3,5,6 -trichloropicolinic acid (picloram).
Picloram promoted the growth of wheat, oat, pea, and tomato explants in vitro (Kefford and Caso, 1966).
Picloram has been successfully applied for callogenesis in date palm (Omar and Novak, 1990) and embryogenesis in pejibaje palm (Valverde et al., 1987). Other plants have also responded to picloram including the following: bamboo
(Huang and Murashige, 1983), sugarcane (Fitch et al., 1983;
Fitch and Moore, 1990), Psoralea bituminosa. Nicotiana tabacum. Salvia mellifera (Goodin and Becher, 1987),
Gasteria and Haworthia (Beyl and Sharma, 1983). 27
In hybrid sugarcane, picloram maintained regeneration ability for more than 12 months, whereas 2,4-D did not
(Fitch and Moore, 1990). Picloram was faster than 2,4-D for callogenesis, embryo induction, and final yield of embryos in Gasteria and Haworthia (Beyl and Sharma, 1983). On the other hand, Saccharum spontaneum clones were slower to form callus (Fitch et al., 1983). More phenolics were produced from cut surfaces of Saccharum spontaneum on picloram compared to 2,4-D. Also some sugarcane hybrids produced callus sooner on 2,4-D than on picloram. However, picloram was utilized to maintain regenerative callus lines over long periods of time (Fitch et al., 1983).
Plant tissues release peroxidases and lAA oxidases into medium. These degrade lAA. Conjugates of lAA protect it from peroxidases and later liberate lAA for hormonal regulation of cell activity (Cohen and Bandurski, 1978; Bonga and Von
Aderkas, 1992). lAA-ala or lAA-asp induced callogenesis and
lAA-asp with KN induced plant regeneration of coconut
embryos (Bhalla-Sarin et al., 1986). lAA-ala stimulated
callus growth, root initiation and inhibited shoots, whereas
lAA-asp stimulated shoot number in tomato leaf culture
(Pence and Caruso, 1984).
The presence of cytokinins, auxins and high concentra
tions of sucrose (0.2 M) stimulated growth of coconut and
date callus (Eeuwens, 1978). Cytokinin at low concentration
together with high concentrations of auxins were necessary 28 for callogenesis and cytokinin at high concentration was necessary for growth of coconut embryos (Bhaskaran, 1985).
However, exogenous cytokinin was unnecessary for initiation of callogenesis of bamboo species (Huang and Murashige,
1983) . Zeatin promoted embryogenesis of carrot cell but 6 - benzylaminopurine (BAP) and kinetin (KN) were inhibitory
(Fujimura and Komamine, 1975).
The role of cytokinins was uncertain in palm regeneration (Brackpool et al., 1986). Low concentration of auxin and KN with high concentration of casein hydrolysate
(CH) and sucrose favored differentiation of embryoids in oil palm (Cui et al., 1984). KN alone was more effective in
inducing differentiation than in combination with lAA and in
the absence of KN callus failed to differentiate since lAA
inhibited the effect of KN (Johri and Bhojwani, 1977).
Srinivasan et al. (1985) successfully induced SE of
Christmas palm with 5 - 50x10'% 2,4-D and 5x10'% BAP, and
plantlet formation occurred on hormone-free medium with
glutamine. However, auxin or cytokinin inhibited SE in
'Shamouti' orange cultures (Kochba and Spegel-Roy, 1977) .
Cytokinins were not necessary for embryogenesis in most
graminous species but might be used in conjunction with 2,4-
D for induction and maintenance of embryogenic calli (Vasil
and Vasil, 1984). Callus was initiated with either BAP or KN
while 2,4-D alone was not effective in longan, BAP was much
less effective for induction of embryogenic competency than 29
KN, although it was more effective in stimulating callus growth (Litz, 1988).
Some cytokinins were important in rapid development of embryoids in oil palm leaf callus (Pannetier et al., 1981).
BAP greatly increased the fresh weight of coconut callus
(Eeuwens, 1978; Kuruvinashetti and Iyer, 1980) and date palm callus (Eeuwens, 1978; Sharma et al., 1984). Among cytokinins, 6 -(7 -7 -dimethylallylamino) purine (2iP) was most active when compared to BAP or KN (Murashige, 1974).
Adenine (Ad) enhanced organ initiation and favored multiplication of diploid cells over endopolyploid cells
(Murashige, 1974). Adenine sulfate (AdS) at 25 mg*l’’ im proved plantlet growth of oil palm (Thomas and Rao, 1985).
Zeatin was the most effective for shoot development of
Actinidia chinensis (Harada, 1975), and at a level of 0.5 mg*!''' increased the number of buds in date culture (Branton and Blake, 1989).
KN at 2xlO'^M was the most effective cytokinin in producing shoot buds with Taxillus vestitus endosperm (Johri and Nag, 1970). Other researchers used a combination of two cytokinins, such as KN and BAP on culture of sandalwood endosperms (Sita et al., 1980), Annona squamosa (Nair et al., 1986), coconut embryos (Gupta et al., 1984; Karunaratne and Periyapperuma, 1989) or three cytokinins, such as AdS,
2iP and BAP on coconut inflorescences (Branton and Blake,
1983a; 1986). 30
After treatment with high auxin levels, transfer to hormone free medium or medium with very low auxin concentration induced embryo germination of callus in oil palm leaves (Rao et al., 1987), Christmas palm embryos
(Srinivasan et al., 1985), date palm embryos (Reynolds and
Murashige, 1979; Zaid and Tisserat, 1984), date palm axillary buds (Tisserat and DeMason, 1980; Sharma et al.,
1984), date palm shoot tips (Sharma et al., 1984), Brachea dulchis. Livistona decipiens. Phoenix pusilla. P. svlvestris. Prostoea sp. and Sabal minor embryos (Zaid and
Tisserat, 1984), Howeia fosteriana and Chamaedorea costaricana embryos (Reynolds and Murashige, 1979) and
Euterpe edulis embryos (Guerra and Handro, 1988).
In coconut, auxins, particularly 2,4-D were found to be essential for embryogenesis, as complete withdrawal of 2,4-D suppressed embryogenesis (Brackpool et al., 1986;
Karunaratne and Periyapperuma, 1989). 2,4-D at 12 - 20x10'^
induced embryogenic callus of 6 - 7 month-old embryos
(diameter 0.2 - 1.5 mm) and plant regeneration occurred when
BAP and KN at lO'^M each were incorporated into the medium containing 2 - 8 xlO'^M 2,4-D (Karunaratne and Periyapperuma,
1989) .
Combination of 2,4-D and BAP induced SE of young and mature coconut leaf (Pannetier and Buffard-Morel, 1982a).
Additionally, lO"'^ M 2,4-D with 5x10'^ BAP, SxlO'^^M 2iP and
27xlO'^M Ads induced embryoids and plantlets from 31 inflorescence tissues of coconut cv. Malayan Dwarf (Branton and Blake, 1983a; 1986).
Gibberelic acid (GAj) was used for germination of somatic embryos in sandalwood (Sita et al., 1980), horse chesnut (Radojevic, 1988) and fostered well-organized shoot and root production of embryonal structures in oil palm callus culture (Nwankwo and Krikorian, 1986).
Antiauxin, i.e. 7-aza-indole (AZI) and 5 hydroxy nitrobenzylbromide (HNB) stimulated embryogenesis in orange cv. Shamouti (Kochba and Spegel-Roy, 1977), whereas B- napthelene acetic acid, phenylpropionic acid, triiodobenzoic acid (TIBA), 2 (p-chlorophenoxy) 2-methylpropionic acid
(PCMP) as well as abscisic acid (ABA) prevented recallusing
of embryoids in soapnut (Sapindus trifoliatus^ (Desay et
al., 1986).
ABA promoted accumulation of storage lipids,
carbohydrates and proteins in somatic embryos (Arnold and
Hakman, 1988; Hakman and Arnorld, 1988; Feirer et al., 1989;
Roberts et al., 1990), supressed abnormal development,
inhibited precocious germinaton and promoted maturation of
somatic embryos in conifer cultures (Durzan and Gupta, 1987;
Boulay et al., 1988; Roberts et al., 1990).
Putrescine is a polyamine, and can be associated with
control of cell growth and division in microbial, animal 32 and plant systems (Audisio et al., 1976; Fienberg et al.,
1984; Feirer et al., 1984; Wu and Kuniyuki, 1985; Evans and
Malmberg, 1989). Polyamines stimulate DNA, RNA, and protein synthesis in plants and animals. An increasing concentration of polyamines is associated with intense mitotic activity in meristematic tissues of tomato and potato (Cohen et al.,
1982) and the initiation of sprouting potato tubers (Kaur-
Sawhney et al., 1982).
Montague et al. (1978) and Bradley et al. (1984) reported that polyamines were involved in cellular differentiation during embryogenesis. Addition of putrescine to the culture medium restored embryogenesis in cultures of wild carrot petiole (Feirer et al., 1984). Putrescine at level O.lxlO'^M - 10‘^M was the most effective polyamine for stimulating division of almond protoplast-derived cells (Wu and Kuniyuki, 1985; Evans and Malmberg, 1989) and increased plant regeneration in apple leaf culture (James et al.,
1988) .
5. Browning
Explant browning is often associated with failure of explant survival. In some adult materials, it may be very
severe, especially in palms which causes the inhibition or 33 cessation of growth (Ventura et al., 1966; Balaga and De
Guzman, 1971; De Guzman et al., 1971; Kuruvinashetti and
Iyer, 1980; Monfort, 1985; Paranjothy, 1986a, 1987;
Krikorian, 1988; Paranjothy et al., 1990). However, explants which have become completely black can provide successful
cultures (Jones, 1974).
Phenolics, tannins or oxidized polyphenols are
synthesized through shikimic acid, phenylpropanoid,
flavonoid and terpenoid pathways. These substances are
abundantly present in some plants and act as inhibitory
agents (Preece and Compton, 1991). Polyphenol is the most
common one (Forrest, 1969; Davies, 1972). Phenolics cause
oxidative browning in explants which leads to discoloration
of the culture medium. Oxidized phenolic compounds are fre
quently exuded into the medium by injured woody tissues
causing lethal browning or blackening of explants (Alderson,
1987).
Immature endosperms were more responsive jji vitro than
mature ones (Cheema and Mehra, 1982). This was partially due
to oxidation products which were more abundant in the older
explants (Sugimura et al., 1988; Preece and Compton, 1991).
Browning can be reduced or eliminated through the use
of liquid media, activated charcoal, and anti-oxidants such
as ascorbic or citric acids, sodium hydrosulfite, cystein,
diethyldithiocarbamate (DTT), potassium ethylxanthate,
thiourea, benzimidazole, sodium bisulfite. 34 polyvinylpyrrolidone (PVP), polyclar, glutathione and bovine serum albumin. Furthermore, more frequent transfers,
incubation with reduced illumination or in complete darkness can also reduce browning. Presoaking explants in sterile water, changing medium, avoiding high temperatures, dis
carding explants which show browning, reducing explant
thickness and choosing the most suitable stage for explants
are important considerations for the minimization of
browning (Murashige, 1974; Reynolds, 1982; Tisserat, 1984;
Pannetier and Buffard-Morel, 1986; Sugimura and Salvana,
1989; Monnier, 1990a; 1990b; Preece and Compton, 1991; Jesty
and Francis, 1992; Krikorian, 1994).
Addition of auxins, particularly 2,4-D at high
concentrations, led to browning of coconut leaf (Pannetier
and Buffard-Morel, 1986). Sugimura and Salvana (1989)
observed that 2,4-D at 2.26xlO'^M - 4.52xlO'^M caused severe
browning of tissues regardless of the stage and size of
coconut inflorescence culture. 2,4-D at levels higher than
30xl0'^M inhibited callusing and enhanced browning of
coconut embryos (Karunaratne and Periyapperuma, 1989).
Callus precociously isolated from explants also caused
browning and necrosis with coconut inflorescences (Verdeil
et al., 1994). Addition of cytokinin, i.e. KN also caused
more browning than auxin in palm cultures (Reynolds, 1982). 35
6 . Culture period
Callus aging prior to subculture increased embryoid development in Citrus sinensis. Six and 14-week-old callus prior to subculture produced 10 times and 100 times, respectively more than three-week-old callus but 2 0 -week-old callus lost totipotency (Kochba and Button, 1974).
Cells in callus increased in ploidy and lose regeneration potential with prolonged culture (Reinert and
Backs, 1968; Johri and Srivastava, 1973; Smith and Street,
1974) . In Citrus limpn, cells declined from 100% diploid to
71% within one month and to 33% by the end of three months
(Murashige et al., 1967).
However, endosperm cultures of rye grass remained triploid after 10 years of culture (Norstog et al., 1969).
In endosperm culture of cucumber, there were abnormal nuclear divisions (Nakajima, 1962), a high degree of polyploidization and various kinds of mitotic
irregularities, such as chromosome bridges and lagging chromosomes in rye grass, maize, Croton. Jatropha and Lolium
(Norstog, 1956; Johri and Bhojwani, 1977).
According to Dutt (1953), the nuclei of coconut endosperms were of varied sizes (diameter from 16.6 to 72.2
/Lt) and chromosome numbers [32 (2n) , 48 (3n), and 160 (lOn)], whereas Abraham and Mathew (1963) observed numbers of 48
(3n), 96 (6n), and 192 (12n). The endosperm callus of parsley consisted primarily of triploid cells but plants 36 derived were predominantly diploid (Masuda et al., 1977).
Regenerating capacity in parsley was retained for one- half year in parsley cultures (Masuda et al., 1977), one year in sandalwood cultures (Sita et al., 1980), two years in Putrani iwa cultures and 30 months in Dendrophthoe and
Taxillus cultures (Johri and Srivastava, 1973; Johri and
Nag, 1974).
Rapidly growing callus (doubling time of 10 to 15 days) of oil palm retained embryonic potentiality after five years of subculture on auxin-containing media (Hanower and
Pannetier, 1982). 37
CHAPTER III
INITIATION OF CALLOGENESIS
INTRODUCTION
Coconut explants, especially mature plants are recalcitrant in vitro. Callus formation (callogenesis) is uncertain, and is influenced by endogenous and exogenous factors such as genotype, physiological maturity, media and disinfestant.
Fisher and Tsai (1978) reported callogenesis occurred in only a single explant of young coconut cv. Golden Malayan
Dwarf endosperm in vitro on White medium. Kumar et al.
(1985) had over 30% callogenesis from endosperm explants with enclosed embryos from 6 - 7 month-old of coconut cv.
West Coast Tall on Eeuwens medium. They also limited the use of disinfestant solution. Bhalla-Sarin and Bagga (1983) failed to get callogenesis from 8 - 1 2 month-old coconut cv.
West Coast Tall, Dwarf and Laccadive.
Coconut endosperm is free of microorganisms (Fernandez,
1988) and an axenic explant can be obtained from the inside of a surface sterilized fruit.
Preliminary experiments showed that coconut endosperm at 7 - 8 months postanthesis (spoon stage) has already developed enough cellular endosperm on the antipodal and 38 micropylar regions, to harvest sufficient explants for experiments. The embryo was not necessary for initiation of callogenesis, and slicing the endosperm inhibited tissue growth. The medium formulation of Branton and Blake (1986) with the addition of putrescine and phytagel produced
earlier and faster callus growth than those of Kumar et al.
(1985) or Fisher and Tsai (1978).
The objective of this chapter is to establish a
protocol for initiation in vitro of callogenesis of coconut
endosperm.
MATERIALS AND METHODS
Two fruits were taken from one coconut tree (Cocos
nucifera) cv. Samoan Dwarf grown in the Magoon greenhouse
facility of the University of Hawaii at Manoa, on January 26
and January 27, 1994. Another two fruits from different
trees grown in Moanalua, Oahu were harvested on April 26,
1994.
Fruits were surface disinfested with 95% ethyl alcohol,
punctured to remove liquid endosperm and cut longitudinally
with a sterile knife. Solid cylindrical endosperm plugs (8
mm in diameter and 2 - 6 mm thick) were aseptically cored
with a cork borer and scooped with a spoon. Explants were
taken from either the micropylar region (upper half of fruit 39 with embryo) or the antipodal region (bottom half of fruit).
The basal medium (BM) of (Branton and Blake, 1986) was modified by addition of 10 mg*l'^ putrescine and
substitution of agar with 1.7 mg*l'^ phytagel (Sigma
Chemical Co. p-8169). Activated charcoal (AC) from Sigma
Chemical Co. c-43 8 6 was used at 2.5 g*r‘ and supplemented with various concentrations of 2,4-dichlorophenoxyacetic
acid (2,4-D) or 4-amino-3,5,6-trichloropicolinic acid
(picloram) at 0, 10'^, 10'^ 10'^, and lO'^M. 6-
benzylaminopurine (BAP) was used in some treatments at 10'
^M.
The pH was adjusted to 5.7 before it was autoclaved.
The medium was poured into 2.5 x 15 cm test tubes (14 ml) or
into 125 ml erlenmeyer flasks (40 ml) and autoclaved at
121°C and 1 kg*cm'^ pressure for 15 minutes. After
autoclaving, the medium was shaken every 10 minutes before
gelling in order to disperse AC.
The medium was stored for about one week as recommended
by Ebert and Taylor (1990), in order to equilibrate growth
regulators in AC. Single explants were placed into test
tubes with the uncut surface [(facing away from the endocarp
(shell)] upright. Each tube was weighed prior to and after
culture. The cultures were incubated at approximately 31°C
in the dark.
The experimental design was a randomized complete block
design (RCBD) with each fruit as a block. Treatments were 40 factorial combinations of endosperm region (micropylar and antipodal), auxin (2,4-D and picloram) and their concentrations (0, IC'^M, lO'^M, lO'^M and lO’^M) and cytokinin
(with or without lO'^M BAP) with 12 replications.
The schedule for subculture was as follows for initiation of callogenesis, callus growth and morphogenesis:
Auxin Transfer
[2,4-D (D) or picloram (P)] [weeks of culture (WOC)]
Initial Trans.1 Trans.2 Trans.3 Trans.4 Trans.5 conc. (9 WOC) (16 WOC) (21 WOC) (26 WOC) (31 WOC) 0 BM BM BMBM BM 10'^M D lO'^M D lO'^M D ± 10‘®M D ± BM BM 10‘^M BAP 10‘^M BAP
lO'^M P 10'^M P lO'^M P ± lO'^M P ± BM BM lO'^M BAP 10‘^M BAP lO'^M D 10'^M D lO'^M D ± 10'®M D ± BMBM 10‘^M BAP lO'^M BAP
lO'^M P 10''^ P lO'^M P ± 10'®M P ± BM BM lO'^M BAP lO'^M BAP 10'^M D 10‘^M D lO'^M D ± 10’®M D ± BM BM lO'^M BAP 10‘^M BAP
10 ■'^M P 10'*^ P 10'^M P ± 10'®M P ± BM BM lO'^M BAP lO’^M BAP 10'^M D 10 ■'^M D lO'^M D ± 10'®M D ± BM BM lO'^M BAP lO'^M BAP
lO'^M P lO'^M P lO'^M P ± 10‘®M P ± BM BM lO'^M BAP lO'^M BAP
Percentage callus formation was computed 31 after WOC,
Data were analyzed with the General Linear Models (GLM) procedure of Statistical Analysis System (SAS). 41
Browning was evaluated visually at every transfer using numerical scores ranging from 0 to 3 (0 = no browning, 1 =
little browning, 2 = medium browning, and 3 = high browning). Data were analyzed with GLM and non parametric one way (Kruskal-Wallis test) procedures of SAS.
RESULTS AND DISCUSSION
Explants formed callus after approximately three WOC.
Callus appearance varied within the control and was similar
to the other treatments (Figure 1). Callus grew
predominantly on the uncut surface of the explant and also
on the side of the endosperm (Figure 2). Eventually, the
callus grew and covered the entire explant. Callus was
initially yellow-white and firm, it then became pale in
color and slightly friable after 21 WOC (Figure 3).
Figure 1 shows the varied growth and development of control
explants after four WOC. Some explants remained rather
white, brownish and swollen while others turned dark brown
and formed callus.
Figure 2 shows initial callus grown from control
cultures after 7 WOC. Callus first appeared on top of the
explant or from the side which touched the medium.
Figure 3 shows callus which had become pale with
friable appearance after 21 WOC. Callogenesis occurred in 42 almost all explants after 31 WOC, including the control.
Average callogenesis was 98.98%, 99.44, 97.92% and 98.98% for fruit 1, 2, 3 and 4, respectively (Table 4).
Callogenesis of antipodal tissues was 98.89%, 98.89%,
97.92% and 100.00% for fruit 1, 2, 3 and 4, respectively
(Table 4). Results with micropylar tissues which was 99.07%,
100.00%, 98.15% and 97.96%, respectively (Table 4). There was no significant difference in callogenesis between the two regions of the endosperm.
Callogenesis of tissues treated with 2,4-D was 98.89%,
100.00%, 96.30% and 98.89% for fruit 1, 2, 3 and 4,
respectively (Table 4). Callogenesis of tissues treated with
picloram which was 99.07%, 98.89%, 99.54% and 99.07% for
fruit 1, 2, 3, and 4, respectively (Table 4). There was no
significant difference in callogenesis between explants
treated with 2,4-D or picloram.
Callogenesis of the control was 100.00%, 100.00%,
97.92% and 100.00% for fruit 1, 2, 3 and 4, respectively
(Table 4). Callogenesis of tissues treated with the 10®M of
both auxins was 100.00% for all fruits (Table 4).
Callogenesis of tissues treated with the 10'^ of both auxins
was 100.00%, 100.00%, 100.00% and 97.92% for fruit 1, 2, 3
and 4, respectively (Table 4). Callogenesis of tissues
treated with the lO^'M of both auxins was 97.50%, 100.00%,
95.83% and 100.00% for fruit 1, 2, 3 and 4, respectively
(Table 4) . Callogenesis of tissues treated with the lO'^M of 43 both auxins was 97.92%, 97.50%, 95.83% and 97.50% for fruit
1, 2, 3 and 4, respectively (Table 4). Consequently, there was no significant difference in callogenesis between different auxins or their concentrations. Furthermore, auxin did not significantly increase callogenesis compared to the hormone-free control.
Callogenesis of tissues treated with the addition of
10-^ BAP which was 98.96%, 100.00%, 95.83% and 98.96% for fruit 1, 2, 3 and 4, respectively (Table 4). Callogenesis without the addition of BAP was 99.00%, 99.00%, 99.58% and
99% for fruit 1, 2, 3 and 4, respectively (Table 4).
Addition of 10'^ BAP did not significantly affect callogenesis.
Tissue (endosperm and callus) browning varied within or among treatments. Figure 4 shows that browning varied from little (I = score 1) to medium (II = score 2) to high (III = score 3) within the control after 21 WOC.
Tissue color changed progressively, from white to tan to brown, to dark brown and to black. Then new yellowish white callus grew from the black callus and this sequence was repeated through many cycles. Figure 5 shows the various colors produced by antipodal tissues treated with 10"^M 2,4-D after 13 months of culture.
Tissue browning by all treatments was slight after 9
WOC (score = 1.18). Tissue browning increased substantially after 16 WOC (score = 1.75) and reached maximal browning 44 after 21 WOC (score = 2.25). Thereafter browning decreased slightly at 26 (score = 2.23) and 31 WOC (score = 2.18)
(Table 6 and Figure 6).
The browning scores of antipodal tissues from all treatments were 1.29, 1.73, 2.27, 2.32 and 2.30 after 9, 16,
21, 26 and 31 WOC, respectively, while those of micropylar tissues were 1.07, 1.76, 2.24, 2.14 and 2.05 after 9, 16,
21, 26 and 31 WOC, respectively (Table 6 and Figure 7).
Statistical analysis showed that antipodal tissues had significantly more browning than micropylar tissues at 9, 26 and 31 WOC but there were no significant differences between them at 16 and 21 WOC (Table 6). Thus, the endosperm region had a significant effect on initial browning but not on long term results.
The browning score of tissues treated with 2,4-D were
1.15, 1.73, 2.22, 2.21 and 2.19 after 9, 16, 21, 26 and 31
WOC, respectively, while those treated with picloram were
1.21, 1.77, 2.29, 2.24 and 2.17 after 9, 16, 21, 26 and 31
WOC, respectively (Table 6 and Figure 8). Thus, there was no significant difference in browning between the 2,4-D and picloram treatments.
After 9 WOC, the browning score of the control (1.60) and tissues initially treated with the lO'^M auxin (1.53) were significantly higher than those of the other auxin concentrations (Table 6 and Figure 9).
After 16 WOC, the browning of tissues initially treated 45 with the lO'^M auxin was the highest while the control was higher than those of tissues treated with other auxins
(Table 6 and Figure 10).
After 21 woe, the tissues initially treated with the
1 0 '^ auxin showed significantly less browning than the
control or other auxin concentrations (Table 6 and Figure
11) .
After 26 WOC, the tissues initially treated with the
lO'^M auxin had a significantly more browning score than the
rest of the treatments (Table 6 and Figure 12).
After 31 WOC, the tissues initially treated with the
lO'^M auxin had a significantly lower browning score (1.94)
than the other treatments (Table 6 and Figure 13). While
lower auxin concentrations retarded browning during the
initial culture period. Their effect diminished over time.
Eventually the highest auxin concentration significantly
reduced browning.
The browning scores of tissues treated with 10‘^M BAP
was 1.43, 2.22, 2.21 and 2.15 on 16, 21, 26 and 31 WOC,
respectively, while those without BAP was 1.96, 2.28, 2.24
and 2.20 after 16, 21, 26 and 31 WOC, respectively (Table 6
and Figure 14). Addition of BAP significantly decreased
browning after 16 WOC but did not affect it significantly
thereafter.
The presence of an embryo in the endosperm explants was
clearly unnecessary for callus initiation in coconut. This 46 contrasts with other report on callogenesis of coconut endosperm (Kumar et al., 1985). Thus, coconut endosperm behaves like endosperms of Actinidia. Croton bonplandianum.
Putraniiva. Citrus qrandis. Citrus sinensis, pear, pecan, sandalwood and walnut (Johri and Bhojwani, 1977; Wang and
Chang, 1978; Cheema and Mehra, 1982; Srivastava, 1982; Nair et al., 1986; Sita, 1987; Zhao, 1988; Mu et al., 1990).
Callus initiation in our studies occurred within three woe. Similar results occurred with endosperm culture of
Exocarpus cupressiformis (Johri and Bhojwani, 1965).
However, endosperm culture of Osvris wiahtiana required 20 weeks to form a callus (Johri and Bhojwani, 1965).
Callogenesis occurred after four weeks in coconut endosperm
(Kumar et al., 1985), one month in embryo explants of
Geonoma qamiova (Dias et al., 1994), while coconut inflorescence cultures required four months for callus initiation (Verdeil et al., 1994). Coconut (Pannetier and
Buffard-Morel, 1982b) and oil palm (Hanover and Pannetier,
1982) leaves also required over two months before callogenesis occurred. These disparities could be due to a number of additional factors which include genotype effects, media differences and disinfestation methods. It is important to note that explants were not directly exposed to any harsh chemicals with our disinfestation protocol.
Coconut endosperm callus became pale in color with slightly friable texture. This was similar to fast-growing 47 oil palm callus (Ahee et al., 1981; Lioret, 1982). In the latter case, this callus morphology was correlated with the development of somatic embryos. In the future, it would be important to make histological observations of coconut endosperm cultures at this stage of callogenesis to see if somatic embryos are present.
The percentage of callogenesis was very high (over 95% for all treatments). Micropylar and antipodal tissues did not differ significantly in callogenesis. This contrasts with Abraham and Mathew (1963) who reported that micropylar region of coconut endosperm was more meristematic than antipodal region. This could have been due to different maturity of fruit and between in vitro and in vivo.
The growth regulator-free control had very high rates of callogenesis, and auxins did not cause significant differences. However, Karunaratne and Periyapperuma (1989) reported 2,4-D at levels higher than 3x10% inhibited callogenesis in coconut embryo culture. This could have been due to the use different explants. Results with other embryogenic systems show that high auxin levels can be used for the production of embryogenic callus and complete plants in loblolly pine, especially when high level of activated charcoal (AC) (2.5 g*l‘^) are used (Gupta and Durzan, 1987).
The AC greatly reduces the effective auxin concentration
(Ebert and Taylor, 1990). So it is difficult to make direct comparisons between these studies. 48
Callogenesis occurred in coconut endosperm without the addition of growth regulators. On the contrary, high auxin concentration was applied for callogenesis in coconut inflorescence cultures (Branton and Blake 1983a; 1986;
Sugimura and Salvana, 1989; Verdeil et al., 1994), coconut endosperm cultures (Kumar et al., 1985), date palm axillary bud and shoot tip cultures (Sharma et al., 1984) and oil palm leaf cultures (Thomas and Rao, 1985). This was attributed to endogenous growth regulators in coconut endosperm which is known to contain cytokinins (Zwar et al.,
1963; Shaw and Srivastava, 1964; Letham, 1968) and gibberellins (Radley and Dear, 1958). Callogenesis also occurred without any addition of growth regulators in parsley (Masuda et al., 1977) and rice endosperms (Bajaj et al., 1980).
Fisher and Tsai (1978) used coconut endosperm explants of various age and got callus from only one explant but could not repeat these results. This could have been due to the disinfestation method. Bhalla-Sarin and Bagga (1983) failed to induce any callus from endosperm explants probably due to the use of older (8 - 1 2 month-old) fruits and also their exposure to disinfestants. Whereas Kumar et al. (1985) succeeded in producing over 30% callogenesis in coconut endosperm by swabbed with cotton wool containing 90% ethanol. Our success in obtaining a very high percentage of callus formation is noteworthy and could have been due to 49 genotypic effects, the use of young explants and their protection from chemical disinfestants as well media
differences, including the addition of putrescine and AC.
With other coconut explants, the percentage of
callogenesis depended on maturity of the tree. Pannetier and
Buffard-Morel (1982b) obtained 50% callogenesis on leaf
explants from young trees and only 20% from adult trees;
whereas Verdeil et al. (1989) obtained 60% - 70%
callogenesis on leaf explants from 5 year-old trees and 30%
- 40% on 15 - 20 year old trees. 45% callogenesis was the
best obtained from inflorescence explants.
With other plant endosperms, callogenesis occurred 85%
in Taxillus vestitus (Nag and Johri, 1971) 29% in Exocarpus
cupressiformis (Johri and Bhojwani, 1965), 24.5% in parsley
(Masuda et al., 1977), 8 - 25% in grapefruit cv. Duncan
(emitter et al., 1990) and 1.60 - 3.74% in Citrus sinensis
cv. Hongjiang (Chen et al., 1990). Thus our results are in
the highest range of successful endosperm callogenesis.
During culture, oxidative browning (brown to black) of
explants or calli was often observed. Phenolic compounds
exude from excised explants. These compounds are oxidized by
peroxidases or polyphenoloxidases, causing browning of both
plant tissue and medium (Compton and Preece, 1988). This
might reduce growth or kill the tissues (Preece and Compton,
1991).
Severe browning of coconut endosperm occurred in our 50 cultures even though young explants which were shielded from disinfestants were used. We also incubated our cultures in the dark, as this condition was shown to prevent browning of
Geonoma qamiova (Dias et al., 1994).
Explant thickness influenced browning. The antipodal explants which were thicker than micropylar showed more browning. Similar results were obtained by Sugimura and
Salvana (1989) with coconut inflorescence explants. Explants
1 mm thick had 32% browning compared to 11% browning with
0.5 mm thick explants.
The two types of auxin did not cause any significant difference in browning of endosperm tissue in this work.
Furthermore, high auxin concentrations (lO’^M 2,4-D or picloram) did not cause more browning than lower concentrations. On the contrary, Fitch et al. (1983) observed that picloram caused more browning than 2,4-D in
Saccharum spontaneum cultures. In addition, Pannetier and
Buffard-Morel (1986), Karunaratne and Periyapperuma (1989) or Sugimura and Salvana (1989) observed that 2,4-D levels higher than 3xlO'^M caused more browning in coconut explants than lower concentrations. Concentrations of 2,4-D or picloram at lO'^M or less reduced tissue browning in our work. Similar results were observed by Phillips and Henshaw
(1977) on Acer pseudoplatanus cell cultures.
Addition of the cytokinin BAP reduced tissue browning at early stage of callogenesis. Reynolds (1982) reported 51 that KN caused browning in palm tissues. It might be useful to test other cytokinins to see if they had a moderating effect on browning.
Severe browning did not check further growth of cultures. A similar result was reported by Jones (1974) in oil palm and Ettinger and Preece (1985) in Rhododendron cultures. Coconut endosperm probably tolerated high level of
2,4-D or picloram due to the presence of AC in the medium and due to dark incubation (Wang and Huang, 1976; Friborg et al., 1978; Tisserat, 1979a; Blake and Eeuwens, 1982;
Ammirato, 1983; Hu and Wang, 1983; Rao et al., 1987;
Sugimura and Salvana, 1989 and Krikorian, 1994). .52
m i f m
4
Fig. 1. Varied growth and development of endosperm after 4 weeks in control. Endosperm tissue was still white, other was brownish and swollen, while others turned dark brown and formed callus.
Fig. 2. Callus first appeared on the top of the explant, another on the side of the explant which touched the medium after 7 weeks in control. 53
Fig. 3. Callus appearances; pale color and slightly friable after 15 months of culture of tissues treated with lO'^M 2,4-D and another one with addition of 10‘^M BAP.
Fig. 4. Tissue (endosperm and callus) browning were varied from little (I = score 1) to medium (II = score 2) and high (III = score 3) in control after 21 weeks. 54
Fig. 5. Varied colors of callus and new yellowish white callus grown on the black callus of antipodal tissue treated with 1 0 ' % 2,4-D and 1 0 ’% BAP after 13 months. 2.4
2 .1--
1.8--
1. 5- CCLU O 1.2 o (/) 0.9-I-
0. 6-
0.3- 0- 9 16 21 26 31 WEEKS OF CULTURE
Fig. 6. Average browning score of all treatments (comparison between all possible combinations and same letters are not significant difference at the 5% level). ui tji 16 21 26 31 WEEKS OF CULTURE
MICROPYLAR ANTIPODAL
Fig. 7. Effect of endosperm region on tissue browning (same letters are not significant difference at the 5% level).
(ji WEEKS OF CULTURE
2,4-D ™ PICLORAM
Fig. 8. Effect of 2,4-D and picloram on tissue browning (same letters are not significant difference at the 5% level). oi -J 10-6M 10-5M 10-4M 10-3M CONCENTRATION
2,4-D PICLORAM
Fig. 9. Effect of 2,4-D and picloram concentrations on tissue browning after 9 weeks of culture (same letters are not significant difference within and * is significant difference among auxin concentrations at the 5% (ji level). 00 10-6M 10-5M 10-4M 10-3M CONCENTRATION
2,4-D PICLORAM
Fig. 10. Effect of 2,4-D and picloram concentrations on tissue browning after 16 weeks of culture (same letters are not significant difference within and * is significant difference among auxin concentrations at the 5% U1 level). VD 10-6M 10-5M 10-4M 10-3M CONCENTRATION
2,4-D PICLORAM
Fig. 11. Effect of 2,4-D and picloram concentrations on tissue browning after 21 weeks of culture (same letters are not significant difference within and * is significant difference among auxin concentrations at the 5% level). cn o a a
a
0 10-6M 10-5M 10-4M 10-3M CONCENTRATION
2,4-D PICLORAM
Fig. 12. Effect of 2,4-D and picloram concentrations on tissue browning after 26 weeks of culture (same letters are not significant difference within and * is significant difference among auxin concentrations at the 5% level). CONCENTRATION
2,4-D PICLORAM
Fig. 13. Effect of 2,4-D and picloram concentrations on tissue browning after 31 weeks of culture (same letters are not significant difference within and * is significant difference among auxin concentrations at the 5% o^ level). to WEEKS OF CULTURE
WITHOUT BAP WITH BAP
Fig. 14. Effect of 10"®M BAP on tissue browning (same letters are not significant difference at the 5% level).
LOCTi 64
CHAPTER IV
CALLUS GROWTH AND MORPHOGENESIS
INTRODUCTION
Solid endosperm of coconut starts to form first in the antipodal region, then spreads gradually to the micropylar region. Therefore, the thickness of endosperm prior to maturity is different in the two regions, thicker in the antipodal than in the micropylar tissue. However, thickness is almost the same when the fruit matures. Endosperm in the micropylar region is more highly meristematic than in the antipodal region (Abraham and Mathew, 1963).
High concentration of growth regulators, especially auxins, can induce callus initiation while decreased concentrations may promote differentiation (Reynolds and
Murashige, 1979; Tisserat and DeMason, 1980; Sharma et al.,
1984; Zaid and Tisserat, 1984; Srinivasan et al., 1985; Rao et al., 1987; Guerra and Handro, 1988). Among auxins, 2,4-D
is the most effective, also picloram is similar properties to 2,4-D which induces callogenesis and promotes differentiation in many plants (Beyl and Sharma, 1983; Fitch et al., 1983; Huang and Murashige, 1983; Goodin and Becher,
1987; Valverde et al., 1987).
Although the role of cytokinin has not been defined, it 65 is commonly added to induce differentiation (Reynolds and
Murashige, 1979; Tisserat and DeMason, 1980; Branton and
Blake, 1983a; Sharma et al., 1984; Srinivasan et al., 1985;
Rao et al., 1987). Among cytokinins, BAP has been applied to promote differentiation in coconut culture (Pannetier and
Buffard-Morel, 1982a; Branton and Blake, 1983a; 1986;
Sugimura et al., 1988; Sugimura and Salvana, 1989).
Endosperm callus of coconut is considered recalcitrant and only a few cases of morphogenesis have been reported. lAA conjugates (lAA-asp, lAA-ala or their combination) have been applied to induce callogenesis and differentiation in coconut embryo culture (Bhalla-Sarin et al., 1986).
Antiauxins (AZI, PCMP, TIBA) as well as ABA have been used to prevent recallusing of embryoids in soapnut (Sapindus trifoliatus) (Desai et al., 1986) and to enhance morphogenesis in pine cultures (Durzan and Gupta, 1987;
Boulay et al., 1988; Roberts et al., 1990). Zeatin has been applied to induce morphogenesis on Actinidia chinensis
(Harada, 1975; Gui et al., 1988) and combinations of NAA,
BAP and GAj on Annona squamosa and cassava cultures (Kartha et al., 1974).
MATERIALS AND METHODS
Materials and methods of chapter IV were as reported in 66 chapter III. For morphogenesis, approximately 1 g of callus from the control was subcultured on basal medium (BM) supplemented with 8.12x10'^ lAA-ala, 6.20x10''^ lAA-asp, combinations of 8.12xlO‘‘^M lAA-ala + 6.20x10'^ lAA-asp,
16.93xlO''^M AZI, 9.32X10'‘^M PCMP, 4. OOxlO’^M TIBA or 7.57 xlO'
^M ABA. Each treatment had 12 replications. Representative
cultures of all treatments were subcultured on BM, BM
supplemented with 4.56x10'^ zeatin or combination of
5.37xlO'^M NAA, 4.44xlO'‘^M BAP and 2.89x10'^ GA3.
Callus growth was computed by subtraction of the
initial tissue fresh weight after from prior to culture
divided by original weight. Growth rate was computed from
callus growth divided by number of weeks in culture. The
data were analyzed with General Linear Model (GLM),
Orthogonal Contrast and Contrast procedures of Statistical
Analysis System (SAS).
RESULTS AND DISCUSSION
Figure 15 shows that fresh weight of endosperms
increased from 0.13 g of original weight to 3.98 g in 9 WOC,
11.29 g in 16 WOC, 21.65 g in 21 WOC, 37.70 g in 26 WOC and
45.67 g in 31 WOC. Growth rate increased from 0.03 g*week'^
in 9 WOC to 0.14 g in 16 WOC, 0.27 g in 21 WOC, 0.43 g in 26 67 woe and decreased to 0.18 g*week‘^ in 31 WOC (Table 23 and
Figure 16).
Growth rate was significantly different depending on the fruit sources. Figure 17 shows growth rate of tissues from four fruits after 31 WOC. Growth rate of fruits 1 and 2 was significantly higher than the rest. Growth rate between fruit 1 and 2 was not significantly different. Fruits 3 and
4 grew slowly and were nearly equal in their overrall growth
(Table 23).
Although the initial weight of micropylar explants
(0.10 g) was different from antipodal explants (0.12 g ) , their growth rates were not significantly different (Figure
18). The growth rate of both types of explants increased
substantially from 9th to 26th WOC and dropped off
afterwards (Table 23).
When treated with 2,4-D or picloram, growth rate
increased substantially from 9th to 26th WOC and then
declined (Table 23 and Figure 19).
Table 23 shows the effect of 2,4-D and picloram
concentration on growth rate of tissues along duration of
cultures. On 9 WOC, growth rate of tissues treated with 10'
2,4-D and picloram was significantly less than that of
the other concentrations (Figure 20). Growth rate of tissues
treated with lO'^M was higher than lO'^M 2,4-D and picloram
on 16th WOC but was no significant difference with other
concentrations (Figure 21). All concentrations had similar 68 growth dynamics in that growth rate increased until 25 WOC
(Figure 22 and 23). Thereafter, the growth rate of cultures initially exposed to 2,4-D and picloram declined, while growth rate of control continued to increase (Figure 24).
Growth rate of tissues treated with and without 1 0 ‘%
BAP followed a similar pattern, increased 26th WOC and then declined (Table 23 and Figure 25). The addition of BAP did not cause any significant difference in growth rate.
Attempts to induce morphogenesis by subculture of
endosperm callus on BM supplemented with lAA-conjugates,
antiauxin, ABA, zeatin or combination of NAA, BAP and GA3 were unsuccessful.
However, an organized structure developed from
endosperm callus derived from antipodal tissue treated with
1 0 ' % picloram after 21 WOC. The explant came from one of
the two fruits taken from a tree at the Magoon greenhouse
facility. Figure 26 shows the "organ" which was elongate,
opaque and contrasted to surrounding yellowish brown callus.
The "organ" grew bigger and more elongate after two months
(Figure 27). However, growth was very slow. It had several
protrusions on its upper surface after 8.5 months (Figure
28) .
No color change was observed upon transfer of the
"organ" to light. The "organ" became triangular after 12
months (Figure 29). Isolation and transfer of this "organ"
to medium with 2.89x10'% GA3 + 5.37X10'% NAA + 4.44X10'% 69
BAP did not speed its growth. After 14 months, its diameter was 2 mm to 9 mm long, and the shape changed from triangular to cylindrical. Figure 30 shows this structure after 14 months. The "organ" was removed after 14 months for histological studies.
Figure 31 shows that similar structure also developed
from callus derived from the previous experiment with fruits
from Moanalua. This callus was initially treated with
207.04xl0*'^M picloram, subcultured to 8.28xlO'^M picloram,
then subcultured to 2.2 6xlO'^M 2,4-D and 5xlO''^M BAP after 17 months of culture.
Callus growth rate was sigmoid and parallel to tissue
browning. This result was also observed in suspension
cultures of carrot (Sugano et al., 1975) and birch cultures
(Welander, 1988). On the contrary, browning of tissue caused
by polyphenol concentration was inversely correlated with
growth rate in the tea plant (Forrest, 1969) and grapevine
in vitro (Yu and Meredith, 1986). This indicates that plants
can have different growth responses to browning.
Growth rate was significantly different among tissues
derived from different trees as well as from the same tree,
especially in the beginning. Genotype, fruit position in the
bunch of a single tree and seasonality may influence explant
response. Three months separated harvest of fruits from
Magoon and Moanalua. Furthermore, the growth conditions were
very different in these locations. Consequently, there are 70 many uncontrolled factors which could account for these differences.
Growth rate was not significantly different for endosperm from different regions of fruit. Morphogenesis occurred with callus derived from the antipodal region. This did not agree with Abraham and Mathew (1963) who observed that endosperm from the micropylar region was more meristematic than from the antipodal region. However, it is impossible to draw firm conclusions about this since only two organized structures were obtained.
The two types of auxin (2,4-D and picloram) did not cause any significant difference in growth rate. On the contrary, Fitch et al. (1983) reported that growth rate of sugarcane hybrids and Saccharum spontaneum cultures treated with 2,4-D was higher than with picloram. On the other hand,
Beyl and Sharma (1983) reported that growth rates of
Gasteria and Haworthia treated with picloram was greater than with 2,4-D. This indicates that different plants can respond differently to various auxins.
The addition of BAP also did not cause any significant difference in growth rate. On the contrary, Eeuwens (1978),
Kuruvinashetti and Iyer (1980) and Sharma et al (1984) found
BAP increased fresh weight of coconut and date palm callus.
This could be due to the use a different explants or to other factors outlined above.
Callogenesis occurred in the control which had a higher 71 growth than other treatments after 31 WOC. Paranjothy and
Rohani (1982), Reynolds (1982) and Paranjothy (1986a; 1987) reported that auxin was necessary for induction of callogenesis in coconut. Auxin was clearly not required in this study.
Morphogenesis from endosperm callus occurred after 21
WOC, a relatively short time, compared to Pannetier and
Buffard-Morel (1982b) who induced embryoids from leaf
cultures of coconut after 6 months of culture. The growth of
this organ was very slow. The "organ" formed several lumps
on the surface which could have been leaf primordia, shoot
apical meristems or somatic embryos.
Morphogenesis in a few cultures out of a large number
of cultures was reported in the following cases. Less than
one out of 1000 developed roots or plantlets with maize
endosperm (LaRue, 1947). One embryo formed out of 452 pear
endosperm + nucellus cultures (Janick, 1982), only one
callus developed from several thousand coconut anther
cultures (Radojevic cited in Kovoor, 1981). One embryo
developed from over 2 0 0 , 0 0 0 coconut anther cultures
(Monfort, 1985; Thanh-Tuyen, 1990), oil palm (Jones, 1974)
and date palm (Tisserat, 1979b).
Variation of culture responses from tree to tree and
batch to batch, were likely a problem in coconut cultures in
this study, was also reported by Blake (1990; 1991), Thanh-
Tuyen (1990) and Rao and Ganapathi (1993). 72
To date, besides the establishment of embryo-derived plant (De Guzman et al., 1983), only one plant derived from culture has survived and been established in the field in the Solomon Islands by Unilever (Smith, 1986; Blake, 1990;
Rao and Ganapathi, 1993).
Picloram was thought to be promising for callogenesis and morphogenesis of coconut endosperm. BAP was not necessary to induce callogenesis or organogenesis in coconut
endosperm culture.
Long-term culture of coconut callus did not regenerate
(Blake, 1990). In preliminary experiments by us, morphogenesis did occur in endosperm calli on medium with picloram after 17 months. Fitch and Moore (1990) maintained
the ability to regenerate in hybrid sugarcane culture over
12 months with picloram but not 2,4-D.
Putrescine, even though a small amount (10 mg*l'') was
used throughout the culture could have influenced
callogenesis and morphogenesis of endosperm cultures.
However, we have no way of knowing this because we did not
have appropriate controls to measure its effect. Phytagel as
a gelling agent possibly may have the potential to
facilitate callogenesis as well as morphogenesis.
Preliminary studies showed that phytagel was equal or
superior to agar.
In all cases, tissue growth increased substantially
with culture duration but the growth rate decreased after 31 73
WOC. This coincided with transfer to growth regulator-free medium and was most likely due to this. Fruit sources greatly influenced growth rate. There were no significant differences in growth rate between micropolar and antipodal tissues, 2,4-D and picloram and with BAP.
The concentration of 2,4-D and picloram slightly
influenced growth rate as 1 0 ‘^M caused significantly less growth after 9 WOC but not after 21 WOC. The control showed
significantly faster growth after 31 WOC compared to
cultures exposed to either auxin.
Morphogenesis occurred on tissues from different trees
treated with low and high levels of picloram after 21 weeks
and 17 months of culture. This suggests that picloram has
the potential to induce morphogenesis of coconut endosperm
callus and may maintain totipotency in long term culture.
Putrescine and phytagel possibly facilitated the
differentiation. Fig. 15. Average tissue growth of all treatments (comparison between all possible combinations and same letters are not significant difference at the 5% level). -J 4^ WEEKS OF CULTURE
Fig. 16. Average growth rate of all treatments (comparison between all possible combinations and same letters are not significant difference at the 5% level). (ji WEEKS OF CULTURE
FRUIT 1 FRUIT 2 3 f r u it 4
Fig. 17. Growth rate of different fruit sources (same letters are not significant difference at the 5% level). a\ UJ lU
cc L iJ Q. O
UJ I- < cr
a:I o
16 21 26 31 WEEKS OF CULTURE
^ MICROPYLAR □ □ ANTIPODAL
Fig. 18. Effect of endosperm region on growth rate (same letters are not significant difference at the 5% level). UJ UJ
cc LU Q. O
UJ I- £
o cc o
16 21 26 WEEKS OF CULTURE
^ 2,4-D PICLORAM
Fig. 19. Effect of 2,4-D and picloram on growth rate (same letters are not significant difference at the 5% level).
00 LU LU
cc UJ Q. o
LU I- < cc
o cc o
10-6M 10-5M 10-4M 10-3M CONCENTRATION
2,4-D PICLORAM
Fig. 20. Effect of 2,4-D and picloram concentrations on growth rate after 9 weeks of culture (same letters are not significant difference within and * is significant difference among auxin concentrations at the 5% vj level). lU Hi
cc LU Q. O
LU H < CC
5 o CCo
10-6M 10-5M 10-4M 10-3M CONCENTRATION
W 2.4-D PICLORAM
Fig. 21. Effect of 2,4-D and picloram concentrations on growth rate after 16 weeks of culture (same letters are not significant difference within and * is significant difference among auxin concentrations at the 5% 03 level). o CONCENTRATION
2,4-D PICLORAM
Fig. 22. Effect of 2,4-D and picloram concentrations on growth rate after 21 weeks of culture (same letters are not significant difference at the 5% level). 00 M UJ UJ 5 cc UJ Q. O
UJ I- < cc
o cc o
10-6M 10-5M 10-4M 10-3M CONCENTRATION
W 2.4-D PICLORAM
Fig. 23. Effect of 2,4-D and picloram concentrations on growth rate after 26 weeks of culture (same letters are not significant difference at the 5% level). 00 to 0.5 a * a 0.45 LU LiJ 0.4 5 cc. 0.35 LU Q. 0.3 O
LU 0.25 I- a a < 0.2 QC 0.15 a 5 0 . 1- O tr o 0.05-
0 0 10-6M 10-5M 10-4M 10-3M CONCENTRATION
2,4-D picloram
Fig. 24. Effect of 2,4-D and picloram concentrations on growth rate after 31 weeks of culture (same letters are not significant difference within and * is significant difference among auxin concentrations at the 5% c» level). w 2 ^ UJ UJ
a: UJ Q. o
UJ I- < a:
o (£ O
16 21 26 WEEKS OF CULTURE
WITHOUT BAP WITH BAP
Fig. 25. Effect of lO ’^M BAP on growth rate (same letters are not significant difference at the 5% level).
00 Fig. 26. The first appearance of a morphogenesis from antipodal tissue treated with 10'^ picloram after 21 weeks of culture. Bar represents 1 mm.
Fig. 27. The "organ" shape became more elongate after 2 months. Bar represents 1 cm. 86
Fig. 28. The "organ" with several lumps on the surface after 8.5 months. Bar represents 3 mm.
Fig. 29. The "organ" became a triangular shape after 12 months. Bar represents 3 mm. Fig. 30. The "organ" became elongate shape after 14 months. Bar represents 1 mm.
Fig. 31. Another morphogenesis occurred from tissue treated with 2 07.04xl0‘‘^M picloram after 17 months. Bar represents 3 mm. 88
CHAPTER V
HISTOLOGICAL STUDY
INTRODUCTION
Before differentiation is visually recognizable, many changes occur at the cellular and tissue levels. Cells can undergo a series of orderly divisions, to form
"promeristemoids" (Villalobos et al., 1985; Flinn et al.,
1988) or meristemoids (Ross et al., 1973; Murashige, 1974).
The latter aggregation consists of small, isodiametric, thin-walled, micro-vacuolated cells, highly basophilic, densely staining nuclei, nucleoli and cytoplasm (Thorpe and
Murashige, 1970; Reinert et al., 1977;). They may also contain numerous starch grains or lipid deposits (Thorpe and
Murashige, 1970; Ross et al., 1973; Villalobos et al., 1985;
Arnold and Hakman, 1988). Meristemoids may develop as embryos, organs or vascular tissues (Reinert et al., 1977;
Thorpe, 1978).
There are two basic types of cells in callus. These may be classified as morphogenic and nonmorphogenic. Morphogenic cells may produce larger organized entities which develop into shoots, roots or embryos. Nonmorphogenic cells may also form large masses but these never produce organs or embryos.
Morphogenic cells have cytological features similar to those 89 described above for meristemoid cells. Nonmorphogenic cells are large and diverse shapes, tend to have large central vacuoles, peripheral cytoplasm, faintly visible nuclei and contain mature chloroplasts with grana. Callus composed by embryogenic cells (EC) are compact, white-pale in color and nodular surface, glossy and mucilaginous; whereas callus composed of nonembryogenic cells (NEC) are granular, friable, smooth, soft, wet, translucent, brownish or green
(Murashige, 1974; Smith and Street, 1974; Reinert et al.,
1977; Durzan and Gupta, 1987; Becwar et al., 1988; Vasil,
1988; Pareddy and Petolino, 1990; Webb and Flinn, 1991).
There are, however, some exceptions, such as the translucent cells in embryogenic conifer calli (Durzan and
Gupta, 1987; Becwar et al., 1988), pale green embryogenic callus in parsley (Masuda et al., 1977) and friable embryogenic callus in Phalaenopsis (Kim, 1994); whereas compact and hard was NEC in Douglas-fir cultures (Durzan and
Gupta, 1987).
Since few large organized structures were obtained, microscopic observations were taken to determine what types of cell growth and organized development occurred in endosperm callus. 90
MATERIALS AND METHODS
Solid endosperm of fresh young coconut (7 months postanthesis) and some calli of the 3 months old were cut with a sharp and clean razor blade inside a petri plate
containing a small volume of glutaraldehyde fixative. Thin tissue sections ( 1 - 2 mm) were placed under vacuum two times for 1 hour each. Tissues were washed three times with
buffer for 10 minutes each and stored in the refrigerator
overnight. Then the tissues were fixed with 2% osmium
tetroxide and washed three times with buffer. Specimens were
dehydrated in a graded series of ethanol (10% to 70%) for 15 minutes each. Specimens were stored overnight in 70% ethanol
in the refrigerator in stoppered vials.
The specimes were dehydraded with graded series of
ethanol (70%, 80%, 90%, 95%, and 100%) in 30 minute steps
and transferred into vials containing 5 ml of 100% ethanol
and 1 ml historesin (HR) and mixed by swirling, capped in
vials and stored overnight at room temperature. HR was added
daily in increasing volume (3 ml, 3 ml, 4 ml), finally in 2
changes of 5 ml of 100% HR.
The embryo-like "organ-like" structure discussed above
derived from endosperm callus, measured 9 mm in diameter and
10 mm in height. It was cut longitudinally into three parts
and fixed immediately in a 50 ml mixture of 1% acrolene, 2%
glutaraldehyde, 2% paraformaldehyde and 0.05M sodium 91 cacodylate buffer at pH 7.6 and placed twice under vacuum for 2 days. The fixative was decanted, the tissue was washed three times with buffer (0.05M cacodylate) for 30 minutes each, and stored in the refrigerator for several days. The
specimens were completely dehydraded and embedded as described above.
The specimens were transferred to peel-away molds,
aluminum pans or beem capsules containing embedding medium.
The containers were closed to exclude oxygen and placed in
vacuum overnight. Polymerized blocks were glued on wood
blocks or plastic rods. These were sectioned with a rotary
microtome at 5 - 10 /Lt. The sections were stained with
toluidine blue, a combination of toluidine blue and acid
fuchsin, Feulgen-fast green or periodic acid Schiff (PAS).
Polymount was added and a cover slip was applied. Water was
used as a mounting fluid in some cases. In these cases, the
edge of the cover slip was sealed with nail polish.
Stains were applied directly to samples taken from
seven month-old liquid cultures. Cell suspensions were
pippeted onto a glass slide. A few drops of potassium
iodide-iodine (IKI) was added and covered with a coverglass.
The specimens were examined and photographed with a Zeiss
photomicroscope. 92
RESULTS AND DISCUSSION
Figure 32 and 33 show that endosperm cells from young coconut fruit are relatively uniform in shape and size.
Nuclei have up to five nucleoli. In culture, the cells vary in shape and size (Figure 34).
Figure 34 shows differential staining of endosperm callus cells by acid fuchsin and toluidine blue. The tissue on the right side of Figure 34 are darker than those to the left. Most of the darkening is associated with the cell walls and peripheral cytoplasm (Figure 34). Acid fuchsin stains most cell components, especially mitochondria.
Toluidine blue was used as a counter stain (Gurr, 1965). The dark areas of cytoplasm have an accumulation of lipid droplets (Figure 35), while cells in the light area of callus have fewer lipid droplets and have more frequent cell divisions (Figure 36).
Division occurred in many planes (Figure 36 and 41).
Some nucleoli appeared long (Figure 36). Figure 37 shows various round cell clusters which have from three to five cells each. Figure 38 shows a linear file of approximately four cells. A structure in Figure 39 resembles a young embryo with suspensor.
Figure 40 show the formation of structures which 93 resemble promeristemoids (Villalobos et al., 1985; Flinn et al., 1988). Promeristemoid was a term proposed by Villalobos et al. (1985) to describe the formation of the organized cluster of six to eight cells in the early stage in vitro of shoot formation by Pinus radiata cultures. Similar structures were observed by Flinn et al., (1988) with P. strobus.
Larger structures called meristemoids were also observed. Figure 41 shows endosperm callus formed a meristemoid that consisted of a nine celled cluster which heavily stain with PAS due to containing carbohydrates
(Gurr, 1965) and over nine celled cluster (Figure 42 and
43) .
Longitudinal section of the "organ-like" structure indicated the presence of central vascular system, a parenchymatous cortex and a dermal layer (figure 44 and 46).
Figure 45 shows stem tip between sheathing base of cotyledon and vascular tissue toward stem tip. Figure 46 shows vascular tissue containing tracheids. Formation of vascular tissue in the "organ-like" structure of endosperm callus is an indication of organogenesis or embryogenesis. Indeed the
"organ-like" structure resembled a zygotic coconut embryo at the advanced stage with sheathing base completely enclosing the stem tip (Haccius and Philip, 1979).
Closer examination of the dermal layer showed that in some areas it contained a meristematic mantle of closely 94 packed cells subtended by a zone of loosely packed cells
(Figures 47 to 54). Many protuberances were visible in the mantle. Some of these resembled early stages of embryo differentiation (Figure 53), while others looked like primordial shoot apices (Figure 54).
The peripheral cells, especially the organized protuberances differed from cells in the adjacent internal region. The mantle cells were smaller, more densely cytoplasmic and had prominent dark nuclei. Similar cells of leaf and hypocotyl cultures in Torenia fournieri and
Anaqallis arvensis were destined to become embryos, buds and shoot apices (Reinert et al., 1977). Figure 54 shows a protuberance which appears to have a one layered tunica which covers a large group of irregularly-arranged cells which could be the corpus. This organization is only found
in shoot apical meristems and leaf primordia. It is
impossible to distinguish between these two possible
structures in this case. However, the tunica corpus of this
"organ-like" was similar to the shoot apex of Phoenix
canariensis and P. dactvlifera (Ball, 1941). It is possible
that early stages of shoot organogenesis occurred in coconut
endosperm callus.
The shape of the suspensors on putative somatic embryos
was slightly different from those of coconut embryos in vivo
but the section shape of the "organ" was similar to the
advanced stage of coconut embryo (Haccius and Philip, 1979). 95
A structure of seven or more celled clusters on coconut endosperm callus (Figure 40 to 43) was similar to promeristemoids which lead to shoot formation with Pinus taeda (Gupta and Durzan, 1987), P. radiata (Villalobos et al., 1985) and P. strobus cultures (Becwar et al., 1988;
Flinn et al., 1988; Webb and Flinn, 1991). 96
Fig. 32. Endosperm cells of young coconut shows relatively uniform in shape and size fruit, stained with toluidine blue. Bar represents 4 /i.
Fig. 33. Nuclei of endosperm cells consists of 4 - 5 nucleoli, unstained. Bar represents 10 fj.. 97
i> V ''■■•■ ^ i r t ^ ' r - \ ' •
■ ' ’ ' F • * ' ■ < w ^ *«A? » #r.^ 7 ;
Fig. 34. Cells of endosperm callus shows dark and light areas with cells vary in shape and size, stained with acid fuchsin and toluidine blue. Bar represents 10 n.
Fig. 35. Enlargement of dark area of Fig. 34. Cells with a lot of lipid droplets. Bar represents 2 ju. 98
%
Fig. 36. Enlargement of light area of Fig. 34. Cells underwent many divisions with few lipid droplets (1) and long nucleoli (n) . Bar represents 2 /x.
Fig. 37. Various round cell clusters with 3 - 5 cells of coconut endosperm treated with 1 0 ‘^M 2,4-D, stained with IKI. Bar represents 4 jU. 99
Fig. 38. Formation of linear four cells structure, stained with acid fuchsin and toluidine blue. Bar represents 20 fj,. Fig. 39. Structure resembles a young embryo with suspensor of coconut endosperm treated with lO'^M 2,4-D, stained with IKI. Bar represents 2 /n. 100
Fig. 40. Formation of promeristemoid with 7 cells cluster, stained with Feulgen-fast green. Bar represents 4 M.
Fig. 41. Formation of meristemoid with 9 cells cluster, showing different types of division, stained with PAS. Bar represents 2 /Lt. 1 0 1
Fig. 42. Meristemoid consisting over 15 cells cluster of coconut endosperm treated with lO'^M 2,4-D and BAP, stained with IKI. Bar represents 4 /i.
Fig. 43. Meristemoid consisting over 20 cells cluster of coconut endosperm treated with 10-5M 2,4-D and BAP, II.stained with Feulgen-fast green. Bar represents 4 102
V - -• --J ■
Fig. 44. Central vascular tissue of "organ-like" structure, showing parenchymatous cortex and dermal layer, stained with toluidine blue. Bar represents 100 jn.
Fig. 45. Sheating base of the cotyledon (s) and vascular tissue (v) toward stem tip (t) of "organ-like" structure, stained with toluidine blue. Bar represents 3 0 ju. 103
Fig. 46. Enlargement of vascular tissue showing tracheids of xylem, stained with toluidine blue. Bar represents 10 M-
Fig. 47. Meristematic periphery of "organ-like" structure, stained with toluidine blue. Bar represents 30 fj.. 104
Fig. 48. Meristematic mantle of "organ-like" structure, stained with toluidine blue. Bar represents 10 ju.
Fig. 49. Protuberances of "organ-like" structure, stained with toluidine blue. Bar represents 10 /x. 105
' 1-,-bL*^ r>»
Fig. 50. Protuberances of "organ-like" structure, stained with toluidine blue. Bar represents 10 jU.
Fig. 51. Protuberances of "organ-like" structure, stained with toluidine blue. Bar represents 10 fj,. 106
Fig. 52. Protuberance of "organ-like" structure, stained with toluidine blue. Bar represents 40 /i.
Fig. 53. Formation of embryoid (e) of "organ-like" structure, stained with toluidine blue. Bar represents 10 ii. 107
Fig. 54. Formation of one layer of tunica (t) and cell group of corpus (c) of "organ-like" structure, stained with toluidine blue. Bar represents 50 fi. 108
CHAPTER VI
GENERAL DISCUSSION
Callogenesis of coconut endosperm started relatively early after about 3 weeks of culture (WOC), compared to 4 weeks for Kumar et al. (1985) or over 2 months for leaf explants of coconut or oil palm (Pannetier and Buffard-
Morel, 1982b; Verdeil et al., 1994).
The percentage of callogenesis was over 95% for all treatments compared to 30% in coconut endosperm for Kumar et al. (1985), 24.5% in parsley endosperm (Masuda et al.,
1977), 8 - 25% for grapefruit endosperm (Gmitter et al.,
1990), 1.60 - 3.74% for orange endosperm (Chen et al.,
1990), 20% for leaf explants of adult coconut plants and 50% for young coconut plants (Pannetier and Buffard-Morel,
1982b), 60% - 70% for leaf explant of young coconut plants and 30% - 40% in adult coconut plants and 45% for inflorescence explants of coconut (Verdeil et al., 1989).
The high rate frequency of callogenesis obtained in by us is probably due to endogenous and exogenous factors.
Endogenous factors include genotype and physiological maturity of coconut fruits (7-8 months postanthesis), whereas exogenous factors include disinfestant contact, culture medium, temperature and dark incubation.
Inclusion of the embryo is not essential for callus 109 initiation in coconut endosperm as reported in other studies of coconut (Kumar et al., 1985) as well as parsley, pear, pecan, sandalwood, walnut, Actinidia. Croton. Putraniiva and
Citrus endosperm cultures (Johri and Bhojwani, 1977; Cheema and Mehra, 1982; Srivastava, 1982; Nair et al., 1986; Wang and Chang, 1978; Zhao, 1988; Mu et al., 1990).
Growth regulators were not necessary for callogenesis, possibly because coconut endosperm contained endogenous PGR, such as cytokinins and gibberellins (Radley and Dear, 1958;
Zwar et al., 1963; Shaw and Srivastava, 1964; Letham, 1968).
This result did not agree with Paranjothy and Rohani (1982),
Reynolds (1982) and Paranjothy (1986a; 1987) who reported auxin was necessary for callogenesis in coconut culture.
Explant and callus browning occurred even with explants
from young fruit, addition of activated charcoal (AC) to the medium and dark incubation. However, Dias et al. (1994)
succeeded in avoiding browning in palm embryo culture of
Geonoma qamiova. 2,4-D and picloram at the 10‘^M level or
lower, as well as lO'^M BAP level reduced browning. Tissue
browning did not completely check tissue growth as reported
in Rhododendron cultures (Ettinger and Preece, 1985).
Tissue fresh weight increased substantially with time
but growth rate decreased after 26 WOC. Thus the growth of
coconut callus followed a sigmoid pattern. Similar result 110 was reported in carrot root and sycamore cell cultures
(Smith and Street, 1974; Phillips and Henshaw, 1977).
Fruit sources greatly influenced the growth response of tissues in culture. This is probably due to genotypic differences. It is well known that the genotype of explants can play a decisive role on iji vitro development (Murashige,
1974). It is also possible that seasonality might have been involved because there was three months difference in the aquisition of fruits.
The growth rate of callus from the endosperm region
(micropylar and antipodal tissues) did not differ significantly, even though antipodal tissues showed more browning than micropylar. This result did not agree with those of Abraham and Mathew (1963) who reported the micropylar region was more meristematic than the antipodal region. Physiological maturity of the coconut fruit might be
a possible factor and they may used fruit at different maturity.
Growth rate with 2,4-D and picloram did not differ
significantly. On the contrary, Fitch et al. (1986) reported
2,4-D produced faster callus growth than picloram in
sugarcane hybrids and Saccharum spontaneum. while Beyl and
Sharma (1983) reported picloram produced faster callus
growth than 2,4-D in Gasteria and Haworthia. However, these
plants are not closely related to coconut and it is not
surprising that our results should differ from others. Ill
Addition of BAP did not cause any significant difference in growth rate. These findings differ from those of Eeuwens (1978), Kuruvinashetti and Iyer (1980) and Sharma et al. (1984) who reported BAP greatly increased fresh weight of coconut and oil palm callus.
2,4-D or picloram at the 10‘^M level inhibited growth at the beginning but not after subculturing to lO'^M. In all cases, calli were transferred to basal medium after 26 weeks of cultures. Growth rate of the control was highest at 31
WOC. Many workers (Reynolds and Murashige, 1979; Tisserat and DeMason, 1980; Blake and Eeuwens, 1982; Branton and
Blake, 1983a; Gupta et al., 1984; Sharma et al., 1984; Zaid and Tisserat, 1984; Kumar et al., 1985; Karunaratne and
Periyapperuma, 1989; Sugimura and Salvana, 1989; Jesty and
Francis, 1992) used high concentrations of auxin to induce callogenesis in palm cultures. However, transfer to a lower concentration may be required for growth or differentiation
(Murashige, 1974; Paranjothy and Rohani, 1982).
Morphogenesis occurred from antipodal tissue derived from fruit which came from the Magoon greenhouse facility of
University of Hawaii at Manoa. This was initially treated with lO'^M picloram and the organized structure appeared after 21 weeks. Another organized structure developed from a fruit taken from Moanalua, Oahu. It was treated with 207.04
X 1 0 ' ^ picloram after 17 months. This indicates that antipodal tissue has the potential to regenerate and that 112 picloram has a potential to induce organogenesis or embryogenesis of coconut endosperm in young and long term culture.
The "organ" was elongate and opaque. This contrasted with the surrounding yellow-brown callus. The growth of this
"organ" was very slow and took about 14 months to reach a size of 9 mm diameter and 10 mm in height. During its development, it became triangular shape and then reverted to a cylindrical shape. Several lumps appeared on its surface.
It superficially resembled a zygotic coconut embryo at this time (Haccius and Philip, 1979).
Histological study showed some cells of the endosperm
suspensions and callus formed structures which resembled promeristemoids, meristemoids, proembryos and embryos with
suspensors (Gupta and Durzan, 1987; Villalobos et al., 1985;
Becwar et al., 1988; Flinn et al., 1988; Webb and Flinn,
1991).
Histological study of the "organ-like" structure showed
a meristematic layer with a dermal layer, cortex-like region
and central vascular tissue. There were many small
protuberances which resembled embryoids and shoots with
tunica corpus organization. The peripheral cells, especially
in the protuberances were small, highly cytoplasmic and had
prominent dark nuclei. These cells were similar to cells
found in other studies Torenia fournieri and Anagallis 113 arvensis which become embryos, buds and shoot apices
(Reinert et al., 1977).
The tunica corpus of this "organ-like" structure consisted of one layer of tunica and a large group of irregularly-arranged cells of corpus which was similar to the structure of the shoot apex of Phoenix canariensis and
P. dactvlifera (Ball, 1941), which are in the same family as coconut. It was likely that shoot organogenesis occurred in the endosperm calli of coconut. This is potentially important because caulogenesis has not been reported yet in coconut endosperm culture. It helps to answer the question of Blake (1990) whether shoot regeneration instead of somatic embryogenesis could occur in coconut culture.
From the preliminary experiment, a similar organ developed after 17 months of culture on the endosperm callus from tissue treated with 207.04xl0'*^M picloram. This tissue was taken from fruit grown in Moanalua. This indicates that tissue from another genotype could produce organized growth after long time in culture.
Antiauxin, ABA, zeatin or combination of NAA, GA and
BAP did not induce morphogenesis of endosperm callus.
Our work has shown that picloram has the potential for inducing differentiation of endosperm callus. Concentration and time of application of picloram are important factors for further study.
Only two occurrences of organized growth were found 114 from over one thousand coconut endosperm cultures. Such low frequencies are not uncommon in recalcitrant tissues. For example, callogenesis occurred in only one of several thousand anthers of coconut (Radojevic cited in Kovoor,
1981). In another study, only one embryo occurred from over
200,000 coconut anthers (Monfort, 1985). A few embryoids occurred out of a large number of oil palm (Jones, 1974) and date palm cultures (Tisserat, 1979b).
Plant regeneration has not been obtained because of the infrequency of organized growth and the long period of time that it takes to get regenerable structures. However, it is the first time organized growth of this type has been reported for coconut endosperm cultures. These results are encouraging and warrant further studies of coconut endosperm culture of coconut because in vitro regeneration has not been reported despite 40 years of work. APPENDIX
Table 3. Analysis for percentage of callogenesis after 31 weeks of culture.
General Linear Models Procedure
Dependent Variable: CALLUS PERCENTAGE (%)
Source DF Sum of Squares F Value Pr > F
Model 18 312.94404550 0.61 0.8829
Error 125 3537.22969825
Corrected Total 143 3850.17374375
R-Square C.V. CALLUS Mean 0.081280 5.382490 98.8310417
Source DF Type I SS F Value Pr > F BLOCK 3 45.25362986 0.53 0.6604 POS 1 0.17430625 0.01 0.9376 AUXIN 1 14.05625069 0.50 0.4823 CONC 1 25.38888951 0.90 0.3454 BA 1 32.34571436 1.14 0.2871 POS*AUXIN 1 0.17430625 0.01 0.9376 CONC*POS 1 9.91567522 0.35 0.5550 CONC*AUXIN 1 0.00000022 0.00 0.9999 CONC*BA 1 53.14056564 1.88 0.1730 POS*BA 1 20.87357511 0.74 0.3921 AUXIN*BA 1 5.33696130 0.19 0.6648 POS*AUXIN*BA 1 27.22611125 0.96 0.3285 CONC*POS*AUXIN 1 0.34282477 0.01 0.9125 CONC*POS*BA 1 1.33163653 0.05 0.8286 CONC*AUXIN*BA 1 35.14826250 1.24 0.2672 H CONC*POS*AUXIN*BA 1 42.23533603 1.49 0.2241 H (J1 Table 4. A summary of the percentage of callogenesis after 31 weeks of culture.
Source Fruit 1 Fruit 2 Fruit 3 Fruit 4
Average 98.98 + 0.71"a 99.44 ± 0.56ay 97.92 + 1.30a 98.98 + 0.71a
Position A 98.89 + 1 .11a 98.89 + 1 .1 1 a 97.69 + 1 .8 8 a 100 . 0 0 + 0 . 0 0 a M 99.07 ± 0.93a 100.00 + 0 .0 0 a 98.15 + 1.85a 97.96 + 1.40a
Auxin 2,4-D 98.89 + 1 .11a 100.00 + 0 . 0 0 a 96.30 + 2.54a 98.89 + 1 .1 1 a pic. 99. 07 + 0.93a 98.89 + 1 .1 1 a 99.54 + 0.46a 99.07 + 0.93a
C one. 0 10 0 . 0 0 + 0 .0 0 a 100 . 0 0 + 0 .0 0 a 97.92 ± 2.08a 100 . 0 0 + 0 .0 0 a IC'^M 10 0 . 0 0 ± 0 .0 0 a 100.00 + 0 . 0 0 a 100.00 ± 0 .00 a 100 . 0 0 + 0 .0 0 a lO'^M 10 0 . 0 0 ± 0 .0 0 a 100.00 + 0 .0 0 a 100.00 + 0 .00 a 97.92 ± 2.08a 10 ■'^M 97.50 + 2.50a 1 0 0 . 0 0 + 0 .0 0 a 95.83 + 4.17a 100.00 ± 0 .0 0 a 1 0 ‘^M 97.92 + 2.08a 97 . 50 ± 2.50a 95.83 + 4.17a 97.50 ± 2 .50a
BAP 0 99.00 ± 1 .00 a 99.00 ± 1 .0 0 a 99.58 ± 0. 42a 99. 00 + 1 . 0 0 a lO'^M 98.96 ± 1.04a 1 00.00 + 0 .0 0 a 95.83 + 2.85a 98.96 + 1. 04a
^Means ± standard error of 12 measurements.
''Means in the same group followed by the same letter in the columns except for the average are not significantly different at the 5% level.
03 Table 5. Analysis for average tissue browning on the duration of culture.
General Linear Models Procedure >: BROWN R A N K (0-3)
Source DF Sum of Squares F Value Pr > F
Model 4 935.92236368 296.83 0.0001
Error 4757 3749.82984128
Corrected Total 4761 4685.75220496
R-Square C.V. BROWN Mean
0.199738 47.82735 1.85636287
P R 0 C E DURE
Wilcoxon Scores (Rank Sums) for Variable BROWN Classified by Variable TRANS
Sum of Expected Std Dev Mean TRANS N Scores Under HO Under HO Score
3 896 802996.0 796992.0 9772.39018 896.200893 4 882 778535.0 784539.0 9772.39018 882.692744 Average Scores were used for Ties Wilcoxon 2-Sample Test (Normal Approximation) (with Continuity Correction of .5)
S= 778535 Z= -.614333 Prob > IZ! = 0.5390 T-Test approx. Significance = 0.5391
Kruskal-Wallis Test (Chi-Square Approximation) CHISQ= 0.37747 DF= 1 Prob > CHISQ= 0.5390 Table 5. Analysis for average tissue browning on the duration of culture (continued).
Sum of Expected Std Dev Mean TRANSN Scores Under HO Under HO Score
4 882 752247.0 747495.0 9290.34701 852.887755 5 812 683418.0 688170.0 9290.34701 841.647783 Average Scores were used for Ties Wilcoxon 2-Sample Test (Normal Approximation) (with Continuity Correction of .5)
S= 683418 Z= -.511445 Prob > j ZI = 0.6090
T-Test approx. Significance = 0.6091
Kruskal-Wallis Test (Chi-Square Approximation) CHISQ= 0.26163 DF= 1 Prob > CHISQ= 0.6090
Sum of Expected Std Dev Mean TRANSN Scores Under HO Under HO Score
2 918 703625.0 794529.0 9871.71829 766.476035 5 812 793690.0 702786.0 9871.71829 977.450739 Average Scores were used for Ties Wilcoxon 2-Sample Test (Normal Approximation) (with Continuity Correction of .5)
I 7 I — S= 793690 Z= 9.20848 Prob > 1^1 - 0.0001
T-Test approx. Significance = 0.0001
Kruskal-Wallis Test (Chi-Square Approximation) CHISQ= 84.797 DF= 1 Prob > CHISQ= 0.0001
03 Table 5. Analysis for average tissue browning on the duration of culture (continued)
Sum of Expected Std Dev Mean TRANSN Scores Under HO Under HO Score
1 1254 1192373.0 1362471.0 13936.0271 950.85566 2 918 1167505.0 997407.0 13936.0271 1271.79194 Average Scores were used for Ties Wilcoxon 2-Sample Test (Normal Approximation) (with Continuity Correction of .5)
I 7 I — S= 1167505 Z= 12.2056 Prob > 1^1 ~ 0.0001
T-Test approx. Significance = 0.0001
Kruskal-Wallis Test (Chi-Square Approximation) CHISQ= 148.98 DF= 1 Prob > CHISQ= 0.0001
Sum of Expected Std Dev Mean TRANS N Scores Under HO Under HO Score
3 896 775520.0 765632.0 9365.44788 865.535714 5 812 683966.0 693854.0 9365.44788 842.322660 Average Scores were used for Ties Wilcoxon 2-Sample Test (Normal Approximation) (with Continuity Correction of .5)
S= 683966 Z= -1.05574 Prob > j Z] = 0.2911
T-Test approx. Significance = 0.2912
Kruskal-Wallis Test (Chi-Square Approximation) CHISQ= 1.1147 DF= 1 Prob > CHISQ= 0.2911
VO Table 5. Analysis for average tissue browning on the duration of culture (continued)
Sum of Expected Std Dev Mean TRANS N Scores Under HO Under HO Score
2 918 715771.0 826659.0 10420.4664 779.70697 4 882 905129.0 794241.0 10420.4664 1026.22336 Average Scores were used for Ties Wilcoxon 2-Sample Test (Normal Approximation) (with Continuity Correction of .5)
S= 905129 Z= 10.6413 Prob > [ZI = 0.0001
T-Test approx. Significance = 0.0001
Kruskal-Wallis Test (Chi-Square Approximation) CHISQ= 113.24 DF= 1 Prob > CHISQ= 0.0001 Table 6. A summary of browning score of tissues on the duration of culture (0 = no browning, 1 = little, 2 = medium and 3 = high).
Source 9 weeks 16 weeks 21 weeks 26 weeks 31 weeks
Average 1.18 ± 0. 0 3 % 1.75 + 0 . 03b^ 2.25 ± 0 . 0 2 a 2.23 + 0 .0 2 a 2 . 18 + 0.03a
Position A 1.29 ± 0.04a 1.73 + 0.05a 2.27 + 0.03a 2.32 ± 0.03a 2 .30 + 0. 04a M 1.07 + 0. 04b 1.76 + 0.04a 2.24 + 0.04a 2.14 + 0.04b 2.05 + 0. 04b
Auxin 2,4-D 1.15 + 0.04a 1.73 + 0. 05a 2.22 + 0.03a 2.21 + 0.04a 2. 19 + 0. 04a pic. 1.21 + 0.04a 1.77 + 0. 04a 2.29 + 0.03a 2.24 + 0.03a 2. 17 + 0. 04a
Cone. 0 1.60 ± 0 . 06a 1.94 + 0 . 06b 2.29 + 0.05a 2.21 + 0.04b 2.40 ± 0.07a 1 0 '% 0.93 + 0.06b 1.47 + 0 . 08c 1.96 + 0.05b 2.25 + 0.04b 2 .21 + 0. 05a 1 0 '% 1.03 + 0.06b 1.58 + 0. 07c 2.39 + 0.05a 2.42 + 0.05a 2.24 + 0 . 06a 1 0 '% 0.95 + 0.06b 1.64 + 0.07c 2.27 + 0.06a 2.09 + 0. 07b 2. 17 ± 0. 07a 1 0 '% 1.53 ± 0.07a 2.15 + 0. 07a 2.36 + 0.05a 2.18 + 0.06b 1.94 + 0. 07b
BAP 0 1.96 ± 0. 04a 2.28 + 0.03a 2.24 + 0.03a 2. 2 0 ± 0.04a 1 0 '% 1.43 + 2.22 + 2.21 + 0.04a 2.15 + 0. 04a 0.05b 0. 04a
%eans ± standard error of 12 measurements.
%eans in the same group followed by the same letter in the columns except for the average are not significantly different at the 5% level (based on a comparison of possible combinations between the average of treatments).
N> Table 7. Analysis for effect of transfer on growth rate.
General Linear Models Procedure
Dependent Variable: WT GRAM
Source DF Sum of Squares F Value Pr > F
Model 4 12.28140562 62.69 0.0001
Error 675 33.05771185
Corrected Total 679 45.33911748
R-Square C.V. WT_GRAM Mean
0.270879 106.5043 0 .20778647
Source DF Type I SS F Value Pr > F
TRANS 4 12.28140562 62.69 0.0001
Contrast DF Contrast SS F Value Pr > F
T1 VS T2+T3+T4+T5 1 5.35684353 109.38 0.0001 T2+T3 VS T4+T5 1 1.12654362 23.00 0 .0001 T2 VS T3 1 1.04135625 21.26 0.0001 T4 VS T5 1 4.75666222 97.13 0.0001 T1 VS T2 1 1.04135625 21.26 0.0001 T3 VS T4 1 4.75666222 97.13 0.0001 Table 8. Analysis of growth rate after 9 weeks of culture.
General Linear Models Procedure
Dependent Variable: WT_GRAM FRESH WEIGHT(GRAM)
Source DF Sum of Squares Pr > F
Model 10 0.04234996 5.35 0.0001
Error 68 0.05386784
Corrected Total 78 0.09621780
R-Square C.V. WT_GRAM Mean
0.440147 48.70364 0.05778949
Source DF Type I SS F Value Pr > F
BLOCK 3 0.03987013 16.78 0.0001 POS 0.00053882 0 . 68 0.4124 AUXIN 0.00070287 0.89 0.3496 CONG 0.00016710 0.21 0.6475 POS*AUXIN 0.00020228 0.26 0.6150 CONC*POS 0.00013974 0.18 0.6758 CONG*AUXIN 0.00048486 0.61 0.4367 CONC*POS*AUXIN 0.00024416 0.31 0.5806 Table 9. Analysis for effect of fruit source on growth rate after 9 weeks of culture.
General Linear Models Procedure
Dependent Variable: WT GRAM FRESH WEIGHT(GRAM)
Source DF Sum of Squares F Value Pr > F
Model 3 0.03987013 17.69 0.0001
Error 75 0.05634767
Corrected Total 78 0.09621780
R-Square C.V. WT_GRAM Mean
0.414374 47.43058 0 .05778949
Source DF Type I SS F Value Pr > F
FRT 3 0.03987013 17.69 0.0001
Contrast DF Contrast SS F Value Pr > F
F1+F2 VS F3+F4 1 0.02299825 30.61 0.0001 FI VS F2 1 0.00336327 4.48 0.0377 F3 VS F4 1 0.01382092 18.40 0.0001 FI VS F3 1 0.03746698 49.87 0.0001 FI VS F4 1 0.00600911 8.00 0.0060 F2 VS F3 1 0.01886295 25.11 0.0001 F2 VS F4 1 0.00039125 0.52 0.4728
.p. Table 10. Analysis for effect of 2,4-D and picloram concentrations (C) on growth rate after 9 weeks of culture.
General Linear Models Procedure
Dependent Variable: WT_GRAM FRESH WEIGHT(GRAM)
Source DF Sum of Squares F Value Pr > F
Model 4 0.02758916 7.44 0-0001
Error 74 0.06862863
Corrected Total 78 0.09621780 R-Square C.V. WT GRAM Mean
0.286737 52.69725 0.05778949
Source DF Type I SS F Value Pr > F
CONC 4 0.02758916 7.44 0 . 0 0 0 1
Contrast DF Contrast SS F Value Pr > F
CO VS C3+C4+C5+C6 1 0.00334743 3.61 0.0613 C3+C4 VS C5+C6 1 0.00750451 8.09 0.0057 C3 VS C4 1 0.01573538 16.97 0. 0 0 0 1 C5 VS C 6 1 0.00084362 0.91 0.3433 CO VS C 6 1 0.00000003 0.00 0.9955 CO VS C5 1 0.00085345 0.92 0.3405 CO VS C4 1 0.00019538 0.21 0.6476 CO VS C3 1 0.01943751 20.96 0. 0 0 0 1 C3 VS C5 1 0.01165051 12.56 0.0007 C3 VS C 6 1 0.01938973 20.91 0. 0 0 0 1 C4 VS C5 1 0.00023912 0.26 0.6131 C4 VS C 6 1 0.00019061 0.21 0.6516 to Ul Table 11. Analysis of growth rate after 16 weeks of culture.
General Linear Models Procedure
Dependent Variable: WT_GRAM FRESH WEIGHT(GRAM)
Source DF Sum of Squares F Value Pr > F
Model 18 0.93309322 12.77 0.0001
Error 125 0.50754324
Corrected Total 143 1.44063646
R-Square C.V. WT_GRAM Mean
0.647695 44.23011 0 .14406667
Source DF Type I SS F Value Pr > F
BLOCK 3 0.86936648 71.37 0. 0 0 0 1 POS 1 0.00332160 0.82 0.3675 AUXIN 1 0.00250333 0.62 0.4338 CONC 1 0.02427858 5.98 0.0159 BA 1 0.00152249 0.37 0.5414 POS*AUXIN 1 0.00651787 1.61 0.2075 CONC*POS 1 0.00146379 0.36 0.5493 CONC*AUXIN 1 0.00122761 0.30 0.5834 CONC*BA 1 0.00000000 0.00 0.9996 POS*BA 1 0.00004600 0.01 0.9154 AUXIN*BA 1 0.00004693 0.01 0.9146 POS*AUXIN*BA 1 0.00446233 1.10 0.2965 CONC*POS*AUXIN 1 0.00605424 1.49 0.2243 CONC*POS*BA 1 0.00030964 0.08 0.7829 CONC*AUXIN*BA 1 0.00060461 0.15 0.7002 CONC*POS*AUXIN*BA 1 tsj 0.01136771 2.80 0.0968 03 Table 12. Analysis for effect of fruit source on growth rate after 16 weeks of culture.
General Linear Models Procedure
Dependent Variable: WT GRAM FRESH WEIGHT(GRAM)
Source DF Sum of Squares F Value Pr > F
Model 3 0.86936648 71.02 0.0001
Error 140 0.57126998
Corrected Total 143 1.44063646
R-Square C.V. WT_GRAM Mean
0.603460 44.33974 0.14406667
Source DF Type I SS F Value Pr > F
FRT 3 0.86936648 71.02 0 . 0 0 0 1
Source DF Type III SS F Value Pr > F
FRT 3 0.86936648 71.02 0 . 0 0 0 1
Contrast DF Contrast SS F Value Pr > F
F1+F2 VS F3+F4 1 0.71008711 174.02 0 . 0 0 0 1 FI VS F2 1 0.00073856 0.18 0.6712 F3 VS F4 1 0.15854081 38.85 0 . 0 0 0 1 FI VS F3 1 0.65371989 160.21 0 . 0 0 0 1 FI VS F4 1 0.16839339 41.27 0 . 0 0 0 1 F2 VS F3 1 0.61051250 149.62 0 . 0 0 0 1 to F2 VS F4 1 0.14682780 35.98 0 . 0 0 0 1 >0 Table 13. Analysis for effect of 2,4-D and picloram concentrations (C) on growth rate after 16 weeks of culture.
General Linear Models Procedure
Dependent Variable: WT_GRAM FRESH WEIGHT(GRAM)
Source DF Sum of Squares F Value Pr > F
Model 4 0.05778758 1.45 0.2201
Error 139 1.38284888
Corrected Total 143 1.44063646 R-Sguare C.V. WT_GRAM Mean
0.040113 69.23353 0 .,14406667
Source DF Type I SS F Value Pr > F
CONC 4 0.05778758 1.45 0 . 2 2 0 1
Contrast DF Contrast SS F Value Pr > F
CO VS C3+C4+C5+C6 0.00266450 0.27 0.6056 C3+C4 VS C5+C6 0.03021497 3 . 04 0.0836 C3 VS C4 0.02338223 2.35 0.1275 C5 VS C 6 0.00152588 0.15 0.6959 CO VS C 6 0.00623070 0.63 0.4301 CO VS C5 0.01228311 1.23 0.2684 CO VS C4 0.00324338 0.33 0.5689 CO VS C3 0.00461067 0.46 0.4971 C3 VS C5 0.04791721 4.82 0.0298 C3 VS C 6 0.03234153 3.25 0.0736 C4 VS C5 0.00435435 0.44 0.5093 M C4 VS C 6 0.00072496 0. 07 0.7876 00 Table 14. Analysis of growth rate after 21 weeks of culture.
General Linear Models Procedure
Dependent Variable: WT_GRAM FRESH WEIGHT(GRAM)
Source DF Sum of Squares F Value Pr > F
Model 18 6.30285409 61.18 0.0001
Error 125 0.71541530
Corrected Total 143 7.01826939
R-Square C.V. WT_(GRAM Mean
0.898064 28.21770 0 .26810347
Source DF Type I SS F Value Pr > F
BLOCK 3 6.23490894 363.13 0. 0 0 0 1 POS 1 0.00585863 1.02 0.3136 AUXIN 1 0.00116793 0.20 0.6522 CONC 1 0.00239390 0.42 0.5190 BA 1 0.00783956 1.37 0.2441 POS*AUXIN 1 0.00200182 0.35 0.5553 CONC*POS 1 0.00036939 0.06 0.7999 CONC*AUXIN 1 0.00067424 0.12 0.7320 CONC*BA 1 0.00047060 0.08 0.7748 POS*BA 1 0.00407002 0.71 0.4007 AUXIN*BA 1 0.00017810 0.03 0.8603 POS*AUXIN*BA 1 0.00000297 0.00 0.9819 CONC*POS*AUXIN 1 0.02755768 4.81 0.0301 CONC*POS*BA 1 0.00357565 0.62 0.4308 CONC*AUXIN*BA 1 0.00103925 0.18 0.6708 1 to CONC*POS*AUXIN*BA 0.01074540 1.88 0.1731 VO Table 15. Analysis for effect of fruit source on growth rate after 21 weeks of culture.
General Linear Models Procedure
Dependent Variable: WT GRAM FRESH WEIGHT(GRAM)
Source DF Sum of Squares F Value Pr > F
Model 3 6.23490894 371.43 0.0001
Error 140 0.78336044
Corrected Total 143 7.01826939
R-Square C.V. WT_GRAM Mean
0.888383 27.90065 0 .26810347
Source DF Type I SS F Value Pr > F
FRT 3 6.23490894 371.43 0.0001
Source DF Type III SS F Value Pr > F
FRT 3 6.23490894 371.43 0.0001
Contrast DF Contrast SS F Value Pr > F
F1+F2 VS F3+F4 6.05213301 1081.62 0.0001 FI VS F2 0.14857609 26.55 0.0001 F3 VS F4 0.03419984 6.11 0.0146 FI VS F3 4.09962523 732.67 0.0001 FI VS F4 3.38494181 604.95 0.0001 F2 VS F3 2.68729608 480.27 0.0001 F2 VS F4 2.11517884 378.02 0.0001 Table 16. Analysis for effect of 2,4-D and picloram concentrations (C) on growth rate after 21 weeks of culture.
General Linear Models Procedure
Dependent Variable: WT GRAM
Source DF Sum of Squares F Value Pr > F
Model 4 0.03919786 0.20 0.9406
Error 139 6.97907153
Corrected Total 143 7.01826939 R-Square C.V. WT_GRAM Mean
0.005585 83.57742 0.26810347
Source DF Type I SS F Value Pr > F
CONC 4 0.03919786 0.20 0.9406
Contrast DF Contrast SS F Value Pr > F CO VS C3+C4+C5+C6 1 0.00095357 0. 02 0.8906 C3+C4 VS C5+C6 1 0.00007488 0.00 0.9693 C3 VS C4 1 0.03664353 0.73 0.3944 C5 VS C6 1 0.00152588 0.03 0.8619 CO VS C6 1 0.00006885 0.00 0.9705 CO VS C5 1 0.00161540 0.03 0.8579 CO VS C4 1 0.01153255 0.23 0.6325 CO VS C3 1 0.00239201 0.05 0.8275 C3 VS C5 1 0.01190827 0.24 0.6270 C3 VS C6 1 0.00490875 0.10 0.7550 C4 VS C5 1 0.00677329 0.13 0.7140 C4 VS C6 1 0.01472886 0.29 0.5889 Table 17. Analysis of growth rate after 26 weeks of culture.
General Linear Models Procedure
Dependent Variable: WT_GRAM FRESH WEIGHT(GRAM)
Source DF Sum of Squares F Value Pr > F
Model 18 19.99872751 65.08 0.0001
Error 125 2.13391013
Corrected Total 143 22.13263764
R-Square C.V. WT_GRAM Mean
0.903585 30.36890 0.43023333
Source DF Type I SS F Value Pr > F
BLOCK 3 19.79017927 386.42 0.0001 POS 1 0.01164241 0.68 0.4105 AUXIN 1 0.00704201 0.41 0.5219 CONC 1 0.00000587 0.00 0.9852 BA 1 0.03821031 2.24 0.1372 POS*AUXIN 1 0.01402251 0.82 0.3665 CONC*POS 1 0.01250917 0.73 0.3936 CONC*AUXIN 1 0.00000090 0.00 0.9942 CONC*BA 1 0.00781517 0.46 0.4999 POS*BA 1 0.00280599 0.16 0.6859 AUXIN*BA 1 0.00062594 0.04 0.8485 POS*AUXIN*BA 1 0.08183149 4.79 0.0304 CONC*POS*AUXIN 1 0.00828902 0.49 0.4872 CONC*POS*BA 1 0.01602825 0.94 0.3344 CONC*AUXIN*BA 1 0.00397960 0.23 0.6301 CONC*POS*AUXIN*BA w 1 0.00373961 0.22 0.6406 to Table 18. Analysis for effect of fruit source on growth rate after 26 weeks of culture.
General Linear Models Procedure
Dependent Variable: WT_GRAM FRESH WEIGHT(GRAM)
Source DF Sum of Squares F Value Pr > F
Model 3 19.79017927 394.26 0.0001
Error 140 2.34245837
Corrected Total 143 22.13263764
R-Square C.V. WT_GRAM Mean
0.894163 30.06546 0.43023333
Source DF Type I SS F Value Pr > F
FRT 3 19.79017927 394.26 0.0001
Source DF Type III SS F Value Pr > F
FRT 3 19.79017927 394.26 0.0001
Contrast DF Contrast SS F Value Pr > F
F1+F2 VS F3+F4 1 19.76350755 1181.19 0.0001 FI VS F2 1 0.02439472 1.46 0.2293 F3 VS F4 1 0.00227700 0.14 0.7128 FI VS F3 1 10.53313202 629.53 0.0001 FI VS F4 1 10.22567402 611.15 0.0001 F2 VS F3 1 9.54371642 570.39 0.0001 F2 VS F4 1 9.25116436 552.91 0.0001 Table 19. Analysis for effect of 2,4-D and picloram concentrations (C) on growth rate after 26 weeks of culture.
General Linear Models Procedure
Dependent Variable: WT GRAM
Source DF Sum of Squares F Value Pr > F
Model 4 0.04466759 0.07 0.9909
Error 139 22.08797005
Corrected Total 143 22.13263764 R-Square C.V. WT_GRAM Mean
0.002018 92.65449 0.43023333
Source DF Type I SS F Value Pr > F
CONC 4 0.04466759 0. 07 0.9909
Contrast DF Contrast SS F Value Pr > F CO VS C3+C4+C5+C6 1 0.00054162 0.00 0.9535 C3+C4 VS C5+C6 1 0.00430940 0.03 0.8694 C3 VS C4 1 0.00981833 0 . 06 0.8041 C5 VS C6 1 0.02999824 0.19 0.6646 CO VS C6 1 0.00517147 0.03 0.8571 CO VS C5 1 0.00483084 0. 03 0.8618 CO VS C4 1 0.00000181 0.00 0.9973 CO VS C3 1 0.00632938 0. 04 0.8421 C3 VS C5 1 0.03332907 0.21 0.6477 C3 VS C6 1 0.00008766 0.00 0.9813 C4 VS C5 1 0.00696808 0. 04 0.8344 C4 VS C6 1 0.00805058 0.05 0.8222 w Table 20. Analysis of growth rate after 31 weeks of culture.
General Linear Models Procedure
Dependent Variable: WT GRAM FRESH WEIGHT(GRAM)
Source DF Sum of Squares F Value Pr > F
Model 18 3.05824230 6.28 0.0001
Error 125 3.38033312
Corrected Total 143 6.43857542
R-Square C.V. WT_GRAM Mean
0.474987 90.10049 0 .18251458
Source DF Type I SS F Value Pr > F
BLOCK 3 1.49355537 18.41 0.0001 POS 1 0.00002678 0.00 0.9749 AUXIN 1 0.00330721 0.12 0.7271 CONC 1 0.95558243 35.34 0.0001 BA 1 0.06700208 2.48 0.1180 POS*AUXIN 1 0.01309690 0.48 0.4878 CONC*POS 1 0.00500493 0.19 0.6678 CONC*AUXIN 1 0.29351937 10.85 0.0013 CONC*BA 1 0.09350959 3.46 0.0653 POS*BA 1 0.01215968 0.45 0.5037 AUXIN*BA 1 0.00220087 0.08 0.7759 POS*AUXIN*BA 1 0.00027541 0.01 0.9198 CONC*POS*AUXIN 1 0.02104024 0.78 0.3794 CONC*POS*BA 1 0.00043161 0.02 0.8997 CONC*AUXIN*BA 1 0.09627214 3.56 0.0615 CONC*POS*AUXIN*BA 1 0.00125771 0.05 0.8296 u ui Table 21. Analysis for effect of fruit source on growth rate after 31 weeks of culture.
General Linear Models Procedure
Dependent Variable: WT_GRAM FRESH WEIGHT(GRAM)
Source DF Sum of Squares F Value Pr > F
Model 3 1.49355537 14.09 0.0001
Error 140 4.94502004
Corrected Total 143 6.43857542
R-Square C.V. WT_GRAM Mean
0.231970 102.9728 0.18251458
Source DF Type I SS F Value Pr > F
FRT 3 1.49355537 14.09 0.0001
Source DF Type III SS FValue Pr > F
FRT 3 1.49355537 14.09 0.0001
Contrast DF Contrast SS F Value Pr > F
F1+F2 VS F3+F4 1 1.45827763 41.29 0.0001 FI VS F2 1 0.01520768 0.43 0.5128 F3 VS F4 1 0.02007006 0.57 0.4522 FI VS F3 1 0.52042102 14.73 0.0002 FI VS F4 1 0.74489149 21.09 0.0001 F2 VS F3 1 0.71355449 20.20 0.0001 w F2 VS F4 1 0.97296600 27.55 0.0001 cn Table 22. Analysis for effect of 2,4-D and picloram concentrations (C) on growth rate after 31 weeks of culture.
General Linear Models Procedure
Dependent Variable: WT_GRAM FRESH WEIGHT(GRAM)
Source DF Sum of Squares F Value Pr > F
Model 4 1.61868442 11.67 0.0001
Error 139 4.81989100
Corrected Total 143 6.43857542 R-Square C.V. WT_GRAM Mean
0.251404 102.0267 0.18251458
Source DF Type I SS F Value Pr > F
CONC 4 1.61868442 11.67 0.0001
Contrast DF Contrast SS F Value Pr > F CO VS C3+C4+C5+C6 1 1.51861057 43.79 0.0001 C3+C4 VS C5+C6 1 0.01966392 0.57 0.4527 C3 VS C4 1 0.07618980 2.20 0.1405 C5 VS C6 1 0.00422013 0.12 0.7277 CO VS C6 1 1.10935700 31.99 0.0001 CO VS C5 1 1.00043708 28.85 0.0001 CO VS C4 1 1.48934399 42.95 0.0001 CO VS C3 1 0.99005157 28.55 0.0001 C3 VS C5 1 0.00004064 0.00 0.9727 C3 VS C6 1 0.00508904 0.15 0.7022 C4 VS C5 1 0.07271112 2.10 0.1498 C4 VS C6 1 0.04189697 1.21 0.2736 w Table 23. A summary of growth rate (g per week) of tissues on the duration of culture.
Source 9 weeks 16 weeks 21 weeks 26 weeks 31 weeks
Average 0. 030±0. 03 9 % 0.14510.102dy 0.26810.221b 0.43010.394a 0.16510.169c Fruit 1 0.084±0.007a 0.21710.009a 0.51810.012a 0.81910.028a 0.26910.045a 2 0.066±0.008b 0.21110.017a 0.42810.021b 0.78210.032a 0.29810.042a 3 0.022±0.003c 0.02710.004c 0.04110.004d 0.05410.004b 0.09910.008b 4 0.060±0.005b 0.12110.009b 0.08510.005c 0.06510.005b 0.06510.006b Position A 0.060±0.006a 0.14910.012a 0.27410.027a 0.43910.047a 0.18210.024a M 0.055±0.005a 0.13910.012a 0.26210.025a 0.42110.046a 0.18310.026a Auxin 2,4-D 0.061±0.006a 0.14810.012a 0.26510.026a 0.43710.046a 0.18710.022a pic. 0.054±0.005a 0.14010.012a 0.27110.027a 0.42310.046a 0.17810.028a Cone. 0 0.071±0.008a 0.13210.018ab 0.26110.056a 0.43610.098a 0. 47310.111a 10'% 0.071±0.008a 0.15610.016ab 0.26310.038a 0.41410.065a 0.15010.028b 10'% 0.060±0.007a 0.16610.015a 0.27310.037a 0.45710.074a 0.16710.023b 10’% 0.06610.010a 0.14910.016ab 0.29410.043a 0.43610.074a 0.09910.011b 10'% 0.02110.003b 0.11110.024 b 0.24610.040a 0.41110.070a 0.16810.021b BAP 0 0.13810.010a 0.26110.024a 0.44410.046a 0.20010.030a 10'% 0.15110.014 0.27710.028 0.41310.046a 0.13610.013a a a
%eans ± standar error of 12 measurements.
%eans in the same group followed by the same letter in the columns except for the average are not significantly different at the 5% level.
W 00 139
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